Compositions and methods for the diagnosis and treatment of ovarian cancers that are associated with reduced smarca4 gene expression or protein function

ABSTRACT

Provided are compositions and methods for the identification and treatment of ovarian cancers, such as small cell ovarian cancers, in particular small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), which ovarian cancers are characterized by reduced SMARCA4 gene expression and/or protein function and, as a consequence, are sensitive to growth and/or survival inhibition by one or more compounds that restore SMARCA4 gene expression and/or protein function.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationsNo. 61/922,710 filed Dec. 31, 2013 and No. 61/973,759 filed Apr. 1, 2014the contents of each of which are incorporated by reference herein.

GOVERNMENT SPONSORED RESEARCH OR DEVELOPMENT

The work described in this disclosure may have been funded in part bygrants from the National Institutes of Health (National CancerInstitute) to the Core facility of the assignee institution: CancerCenter Support Grant P30 CA008748. The U.S. government may have certainrights in this disclosure.

SEQUENCE LISTING

The present application includes a Sequence Listing in electronic formas a txt file in ASCII format titled “Sequence_Listing_60009_0004WPU1”also bearing the designation 8988979_1.txt and having a size of 83.139kb. The contents of this txt file are incorporated by reference herein.

BACKGROUND OF THE DISCLOSURE

1. Technical Field

The present disclosure relates, generally, to the diagnosis andtreatment of ovarian cancers, such as small cell ovarian cancers, inparticular small cell carcinoma of the ovary, hypercalcemic type(SCCOHT), which ovarian cancers are characterized by reduced SMARCA4gene expression and/or protein function and, as a consequence, aresensitive to growth and/or survival inhibition by one or more compoundsthat restore SMARCA4 gene expression and/or protein function (such asdrugs that target DNA repair pathways) and/or inhibit growthdysregulating and survival promoting consequences of such aberrantSMARCA4 gene expression such as EZH2 hyperactivity.

2. Description of the Related Art

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), is arare, aggressive form of ovarian cancer diagnosed in young women. SCCOHTis generally fatal when spread beyond the ovary. SCCOHT represents lessthan 1% of all ovarian cancer diagnoses, with less than 300 casesreported in the literature to date. Estel et al., Arch Gynecol Obstet284:1277-82 (2011) and Young et al., Am J Surg Pathol 18:1102-16 (1994).The mean age at diagnosis is 23 years and, unlike patients with the morecommon types of ovarian cancer, the majority of these women present withearly-stage disease. Harrison et al., Gynecol Oncol 100:233-8 (2006).Nonetheless, most patients relapse and die within 2 years of diagnosis,regardless of stage, with a long-term survival rate of only 33%, evenwhen disease is confined to the ovary at diagnosis. Seidman, GynecolOncol 59:283-7 (1995). There are no reliable adjuvant treatments thatimprove outcome, but multi-compound chemotherapy is thought to extendsurvival. Estel et al., Arch Gynecol Obstet 284:1277-82 (2011) andPautier et al., Ann Oncol 18:1985-9 (2007).

The tissue of origin remains speculative, and SCCOHT is stillcategorized as a miscellaneous tumor by the World Health Organization.Most tumors are unilateral, and size greater than 10 cm may beprognostically favorable due to earlier onset of symptoms resulting instage migration. Estel et al., Arch Gynecol Obstet 284:1277-82 (2011).Histologic classification can be challenging, but commonly expressedimmunohistochemical markers such as CD10, WT1, and calretinin can beuseful in conjunction with loss of detectable inhibin, S100, andchromogranin expression to exclude histological mimics. McCluggage, AdvAnat Pathol 11:288-96 (2004).

SUMMARY OF THE DISCLOSURE

The present disclosure is based upon the discovery that mutations in theSMARCA4 gene and/or associated regulatory sequences, including certaingermline or somatic mutations, in particular mutations in both allelesof the SMARCA4 gene and/or associated regulatory sequences (i.e.,bi-allelic mutations), which reduce or eliminate SMARCA4 gene expressionand/or that reduce or eliminate SMARCA4 protein levels and/or proteinfunction, are causally linked to certain cancers, including ovariancancers, such as small cell carcinomas of the ovary (SCCO), inparticular SCCO hypercalcemic type (SCCOHT), which is a rare, highlyaggressive form of ovarian cancer.

It was further discovered that restoring SMARCA4 gene expression and/orSMARCA4 protein levels or functionality in SMARCA4-negative cells, inparticular in SCCOHT cells, suppresses cell growth while reducingSMARCA4 gene expression and/or SMARCA4 protein levels of functionalityin SMARCA4-positive cells promotes cell growth.

Based upon these and other discoveries, which are described in detailherein, in certain embodiments, the present disclosure provides methodsfor the diagnosis of cancers, including ovarian cancers, such as smallcell carcinomas of the ovary (SCCO), in particular SCCO hypercalcemictype (SCCOHT), which cancers are associated with reduced or undetectableSMARCA4 gene expression and/or with reduced or undetectable SMARCA4protein levels and/or protein function, which methods include thedetection of one or more mutations, including one or more germline orsomatic mutations, in the SMARCA4 gene and/or associated regulatorysequences in particular mutations in both alleles of the SMARCA4 geneand/or associated regulatory sequences (i.e., bi-allelic mutations),which are known, predicted, or demonstrated to reduce or eliminateSMARCA4 gene expression and/or known, predicted, or demonstrated toreduce or eliminate SMARCA4 protein levels and/or protein function.

In other embodiments, the present disclosure provides methods forinhibiting the growth of cancer cells and for the treatment of cancers,including ovarian cancers, such as small cell carcinomas of the ovary(SCCO), in particular SCCO hypercalcemic type (SCCOHT), which cancersare associated with a cancer cell exhibiting reduced or undetectableSMARCA4 gene expression and/or with reduced or undetectable SMARCA4protein levels and/or protein function, which methods include contactinga cancer cell with, or administering to a cancer patient, one or morecompounds, including one or more polynucleotides, polypeptides, and/orsmall molecules that can restore SMARCA4 gene expression and/or SMARCA4protein levels and/or protein function and, thereby, slow or stop thegrowth of the cancer cell. Alternatively, drugs that target DNA repairpathways and/or inhibit growth dysregulating and survival promotingconsequences of such aberrant SMARCA4 gene expression such as EZH2hyperactivity can be used for treating tumors characterized by reducedor eliminated SMARCA4 expression and/or protein levels or function.

In still further embodiments, the present disclosure provides compoundsand compositions, including pharmaceutical compositions, containingthose compounds, which compounds and compositions may be advantageouslyemployed in the presently-disclosed methods for inhibiting the growth ofcancer cells and for the treatment of cancers, including ovariancancers, such as small cell carcinomas of the ovary (SCCO), inparticular SCCO hypercalcemic type (SCCOHT), which cancers areassociated with a cancer cell exhibiting reduced or undetectable SMARCA4gene expression and/or with reduced or undetectable SMARCA4 proteinlevels and/or protein function. Exemplified herein are compounds,including polynucleotides, polypeptides, and small molecules that can beused to restore SMARCA4 gene expression and/or function and/or restoreSMARCA4 protein level and/or function and, thereby, slow or stop thegrowth of a cancer cell. Alternatively, drugs that target DNA repairpathways and/or inhibit growth dysregulating and survival promotingconsequences of such aberrant SMARCA4 gene expression such as EZH2hyperactivity can be used for treating tumors characterized by reducedor eliminated SMARCA4 expression and/or protein levels or function.These therapeutic approaches may be combined with chemotherapeutic drugsand more specifically drugs that damage genomic DNA in rapidly dividingcells.

In yet other embodiments, the present disclosure provides diagnostickits for identifying a cancer cell or for detecting in a patient acancer cell that exhibits reduced or eliminated SMARCA4 gene expressionand/or function and/or reduced or absent SMARCA4 protein level and/orfunctionality, which diagnostic kits contain one or more reagents thatcan be used alone or in combination to: (a) detect a mutation in aSMARCA4 gene and/or mRNA, such as an insertion mutation, a deletionmutation, a frame shift mutation, a splice site mutation, and a pointmutation, in particular a nonsense mutation and/or a missense mutationin a SMARCA4 gene and/or mRNA; (b) detect a reduction in SMARCA4 mRNAlevel; (c) detect a reduction in SMARCA4 protein level; and/or (d)detect a reduction in SMARCA4 protein functionality.

Within related aspects, the present disclosure also provides diagnostickits that can be advantageously employed in the methods of the presentdisclosure for identifying a cancer cell or a detecting in a patient acancer cell that exhibits reduced or eliminated SMARCA4 gene expressionand/or function and/or reduced or absent SMARCA4 protein level and/orfunctionality, which diagnostic kits contain one or more agents that canbe used alone or in combination to: (a) detect a mutation in a SMARCA4gene and/or mRNA, such as an insertion mutation, a deletion mutation, aframe shift mutation, a splice site mutation, and a point mutation, inparticular nonsense mutation and/or a missense mutation in a SMARCA4gene and/or mRNA; (b) detect a reduction in SMARCA4 mRNA level; (c)detect a reduction in SMARCA4 protein level; and/or (d) detect areduction in SMARCA4 protein functionality. Such reagents may includefor example amplification primers for all or for one or more portions ofthe genomic sequence of SMARCA4 and detectable labels as well aspositive and negative controls wherein said controls are not substancesfound in nature.

These and other aspects of the present disclosure will be bestunderstood in conjunction with the following drawings, which exemplifycertain aspects of the various embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagram showing the position of 11 of 12 SMARCA4 mutationsidentified in SCCOHT and TCGA (The Cancer Genome Atlas) tissue samplesrelative to the domain structure of SMARCA4 protein (Uniprot ID:SMCA4_HUMAN). (Case number 103, which exhibited an exon deletion, is notshown).

FIG. 1B is a bar graph showing the percentages of samples withnon-synonymous SMARCA4 mutations in the SCCOHT and TCGA non-hypermutatedsamples (numbers of samples per study in parentheses). The darker bars(bar corresponding to case No. 12 and upper portions of two-tone bars)represent samples with missense-only mutations, and the lighter bars(bars corresponding to case Nos 100, 231, 34, 35, 275 and 456 and 402 aswell as the lower portion of two-tone bars) represent samples withnon-missense (including nonsense, frameshift, splice site, and indel)mutations.

FIG. 2A is a Sanger sequence trace chromatogram showing analysis of anexemplary SMARCA4 mutation, which, in this case (No. 102) was a singlenucleotide (G>A) somatic mutation within the intron 27 donor splice siteregion. Similar analyses were performed for each of the case Nos 101 and103-112 (data not shown).

FIG. 2B is an immunoblot with an anti-SMARCA4 N-terminal antibody. Ahigh-grade serous ovarian cancer cell line (PEO4) and frozen tumorsamples from 2 patients with high-grade serous ovarian cancer (HGOC#1and HGOC#2) were used as positive controls and show retained proteinexpression. Protein extracted from H1299 non-small cell lung cancercells, deficient in SMARCA4, served as a negative control. Proteinextracted from cases 101 and 102 (SCCO #101 and SCCO #102), each with adonor site splice site mutation, exhibit loss of SMARCA4 proteinexpression.

FIG. 2C is an immunohistochemistry (IHC) image showing loss of proteinexpression from archival SCCOHT tissue stained with a polyclonal SMARCA4antibody. The intense staining of blood vessels and stromal cell nucleiserve as internal controls.

FIG. 3 is a scatterplot with grouped data showing SMARCA4 geneexpression across TCGA tumors for cases with available mutation and RNAsequence data (RSEM). A correlation is seen between inactivating SMARCA4mutations and decreased gene expression across various solid tumors. Atwo-sample Student's t-test comparing samples with non-missensemutations and other samples without mutations or with only missensemutations. For all TCGA samples, the mean RSEM (2050, std: 1760) wasless in samples with non-missense mutations than other samples withoutmutations or with only missense mutations (3724, std: 1692; P=8.7×10⁻⁴).For TCGA lung adenocarcinoma samples, the mean RSEM (601, std: 370) wasless in samples with non-missense mutations than other samples withoutmutations or with only missense mutations (3330, std: 1524; P=2×10⁻⁸).

FIG. 4 is an immunohistochemistry (IHC) image showing results of SMARCA4staining in SCCOHT cases. High-grade serous ovarian carcinoma is used asa positive control. Case numbers are indicated in each panel. Note theintense staining of blood vessels and stromal cell nuclei as internalcontrols.

FIG. 5A is a digital image of a DNA gel and a diagram showing thatone-step RT-PCR confirms that tumor tissue from case 103, which exhibitsa deletion of exons 25 and 26, yields a single band with primers thatspan exons 24 and 27 (*denotes a nonspecific band).

FIG. 5B is a digital image of a DNA gel and a diagram illustrating thatone-step RT-PCR with primers targeting regions upstream and downstreamfrom the deletion site in tumor tissue from case 103 show equalexpression demonstrating continuation of transcription downstream fromthe deletion.

FIG. 6A is a digital image of a DNA gel and a diagram showing analysisof splice site variant in tumor tissue from case 102. One-step RT-PCRconfirms that the exon/intron band is preferentially expressed over theexon/exon band in tumor tissue. The exon-exon primers detected weakerbands, reflecting loss of expression in tumor tissues compared withnormal tissues in cases with splice site mutations. Immunoblots showingmuch reduced SMARCA4 protein levels are shown in FIG. 2B. Theexon-intron primers demonstrated equivalent to greater expression ofretained intron in the tumor tissues. Since SMARCA4 introns may beretained in non-cancer tissues, some intronic expression is expected innormal tissues. These data taken together indicate preferential intronicexpression, as expected, in cDNA sequenced from tumor samples withsplice site mutations.

FIG. 6B is a digital image of a DNA gel and a diagram showing thatone-step RT-PCR with primers targeting regions upstream and downstreamfrom the mutation site in tumor tissue from case 102 show equalexpression demonstrating continuation of transcription downstream fromthe mutation.

FIG. 7A is a digital image of immunoblots showing SMARCA4over-expression in H1299 cells. Representative immunoblots from threebiologic replicates demonstrate a correlation between increased SMARCA4and p21 expression.

FIG. 7B is a bar graph showing cell growth assessment in H1299 cellsover-expressing SMARCA4. Mean cell number counts from three biologicreplicates are shown.

FIG. 7C is a digital image showing representative immunoblot confirmingSMARCA4 knock-down in 293T cells using shRNA. As a control, shNTC(Non-Targeting Control) was used.

FIG. 7D is a line graph showing results from XTT proliferation assay in293T cells depleted of SMARCA4. Means represent three independentexperiments.

FIG. 8 is a Kaplan-Meier survival plot showing overall survival amonglung adenocarcinoma TCGA cases based on SMARCA4 mutations. Medianoverall survival was 11.6 months among 6 patients with inactivatingSMARCA4 mutations compared with 44.6 months for 197 patients withoutinactivating mutations.

FIG. 9 is a sequence map and a Sanger chromatograph showing sequenceanalyses for SMARCA4 in H1299 cell line. An electropherogram from Sangersequencing of genomic DNA validating a 69 nucleotide deletion in theopen reading frame of this control cell line that results in loss ofprotein expression, as shown in FIG. 2B.

FIG. 10 is an immunohistochemistry (IHC) image showing histopathologicalfeatures of a small cell carcinoma of the ovary, hypercalcemic type(SCCOHT). The typical histopathological features of SCCOHT, including acombination of small neoplastic cells forming a pseudofollicular spaceand larger rhabdoid cells, are visible in a sample obtained from 1 of 12tumors that were subjected to target capture and massively parallel DNAsequencing (staining with hematoxylin and eosin).

FIG. 11A is a western blot confirming SMARCA4 knock-down (β-Actin is aloading control and shNTC is a non-targeting control).

FIG. 11B is a graph of relative cell proliferation as a function oftime. These data demonstrate that depletion of SMARCA4 in T80 cellsincreases cell growth.

FIG. 12A is a digital photograph of a western blot confirming SMARCA4knock-down in SMARCA3 depleted 293T cells. β-Actin was used as a loadingcontrol.

FIG. 12B is a plot of tumor volume versus time from xenograft injectionand thus depicting tumor growth in mice xenografted with SMARCA4knock-down in 293T cells (squares) compared to nontargeted control (NTC,diamonds).

DETAILED DESCRIPTION

The present disclosure is based upon the discovery that mutations in theSMARCA4 gene and/or associated regulatory sequences, includingbi-allelic mutations, that reduce SMARCA4 gene expression and/or thatreduce SMARCA4 protein levels and/or function, are very common incertain cancers including small cell carcinoma of the ovary (SCCO), inparticular SCCO hypercalcemic type (SCCOHT), which is a rare, highlyaggressive form of ovarian cancer.

Moreover, as disclosed herein, it was found that restoration of SMARCA4function in SMARCA4-deficient cells suppresses cell growth while loss ofSMARCA4 function in normal cells promotes cell proliferation. Thus,mutations in the SMARCA4 gene, including bi-allelic mutations thatreduce SMARCA4 gene expression and/or that reduce SMARCA4 protein levelsand/or function, are diagnostic of SCCOHT disease phenotype in patientand are predictive of the therapeutic efficacy of a treatment regimenfor SCCOHT that achieve at least a partial restoration of SMARCA4 geneexpression and/or of SMARCA4 protein levels and/or function. Moreover,at least partial restoration of SMARCA4 expression and in any eventfunction presents itself as a therapeutic goal.

In studies leading to the presently-disclosed discoveries the geneticbasis for SCCOHT was determined by sequencing the protein-coding exonsin 279 cancer-related genes in 12 paired SCCOHT tumor and non-tumorsamples. Among those 279 genes, SMARCA4 was the only gene that exhibitedmutations in every tumor sample tested (FIG. 1B) while only four othernon-recurrent somatic mutations were identified in the 278 othercancer-related genes from those 12 SCCOHT samples that were sequenced.In contrast, analysis of 4,784 non-hypermutated tumors across The CancerGenome Atlas (TCGA) revealed somatic mutations in an average of 4.3 ofthose 279 genes (STD 4.4) per tumor. TCGA samples with inactivatingSMARCA4 mutations had more mutations in the other 278 genes sequenced(mean=14) in contrast to the SCCOHT cases.

As discussed herein, the probability of identifying SMARCA4 mutations inall 12 SCCOHT samples is less than 2.22×10⁻¹⁶. Based, in part, upon thistight association between certain bi-allelic, SMARCA4 gene mutations andpatient samples exhibiting the SCCOHT phenotype, the present disclosureprovides methods for the diagnosis of SCCOHT, which methods include, forexample, the detection of reduced SMARCA4 gene expression and/or ofreduced SMARCA4 protein levels and/or functionality, as well as methodsfor the treatment of SCCOHT. The latter methods include at least thepartial restoration of wild-type SMARCA4 gene expression and/orfunction; at least the partial restoration of wild-type SMARCA4 proteinlevels and/or functionality; and/or inhibition of an activity that iselevated with reduced SMARCA4 activity or stimulation of an activitythat is reduced with reduced SMARCA4 activity.

The SMARCA4 mutations that were identified in the 12 SCCOHT tumorsamples are presented herein in Table 1. Based upon the predictedchanges in protein structure and/or observed reduction in protein levelsresulting from those SMARCA4 gene mutations; the high frequencyoccurrence of bi-allelic SMARCA4 mutations; and the tight associationbetween those SMARCA4 mutations and the SCCOHT phenotype, it wasdiscovered that such mutations, which can include insertion mutations,deletion mutations, frame shift mutations, splice site mutations, andpoint mutations, in particular nonsense mutations and/or missensemutations, in the SMARCA4 gene and/or in one or more sequences thatcontrol the expression and/or functionality of the SMARCA4 gene, whichmutations reduce or eliminate SMARCA4 gene expression and/or functionand/or result in reduced or undetectable SMARCA4 protein levels and/orfunctionality, are causative of the SCCOHT phenotype.

TABLE 1 Summary of Patient Characteristics and SMARCA4 Mutations Age atTumor Tumor Case diagnosis Year of Coding sequence Predicted sequenceallele Affected Functional IHC No. (years) diagnosis change proteinchange Variant class reads frequency exon domain# result 101 40 2003 G >A p.Q1182_splice Splice site 1438 0.83 24 Helicase Loss 102 22 2009 G >A p.K1390_splice Splice site 786 0.77 27 Loss homozygous 103 19 2010deletion Exon deletion NA NA 25-26 Helicase Retained 104 21 1998 G > Ap.K587_splice Splice site 198 0.31 9 N/A C > T p.R978* Nonsense 322 0.3419 SNF2 105 25 2009 C > T p.Q331* Nonsense 175 0.5 5 N/A TC > T p.I542fsFrameshift 496 0.48 9 deletion 106 40 2012 C > T p.R1093* Nonsense 6080.47 6 Loss TC > T p.L388fs Frameshift 520 0.47 23 Helicase deletion 10718 2010 GACGAGACCGT p.ETVN1300del In frame 393 0.83 27 N/A CA > Gdeletion 108 22 2011 C > T p.Q847* Nonsense 126 0.94 17 SNF2 Loss 109 322010 T > TG p.L762fs Frameshift 299 0.12 15 SNF2 Loss insertion G > Tp.G836* Nonsense 413 0.49 17 SNF2 110 42 2011 C > T p.Q1166* Nonsense518 0.8 24 Helicase Loss 111 35 2012 C > T p.R1005* Nonsense 609 0.93 20SNF2 Loss germline{circumflex over ( )} 112 28 2011 G > A p.K953_spliceSplice site 288 0.75 18 SNF2 Equivocal

Based upon these and other discoveries, which are described in detailherein, the present disclosure provides:

1. Methods for the diagnosis of cancers, including ovarian cancers, suchas small cell carcinomas of the ovary (SCCO), in particular SCCOhypercalcemic type (SCCOHT), which cancers are associated with reducedor undetectable SMARCA4 gene expression and/or with reduced orundetectable SMARCA4 protein levels and/or protein function, whichmethods include the detection of one or more mutations, including one ormore nonsilent germline or somatic mutations, in the SMARCA4 gene and/orassociated regulatory sequences in particular (but not limited to)mutations in both alleles of the SMARCA4 gene and/or associatedregulatory sequences (i.e., bi-allelic mutations), which are known,predicted, or demonstrated to reduce or eliminate SMARCA4 geneexpression and/or known, predicted, or demonstrated to reduce oreliminate SMARCA4 protein levels and/or protein function;

2. Methods for inhibiting the growth of cancer cells and for thetreatment of cancers, including ovarian cancers, such as small cellcarcinomas of the ovary (SCCO), in particular SCCO hypercalcemic type(SCCOHT), which cancers are associated with a cancer cell exhibitingreduced or undetectable SMARCA4 gene expression and/or with reduced orundetectable SMARCA4 protein levels and/or protein function, whichmethods include contacting a cancer cell or administering to a cancerpatient one or more compounds, including one or more polynucleotides,polypeptides, and/or small molecules that can restore SMARCA4 geneexpression and/or SMARCA4 protein levels and/or protein function and,thereby, slow or stop the growth of the cancer cell;

3. Compounds and compositions, including pharmaceutical compositions,containing those compounds, which compounds and compositions may beadvantageously employed in the presently-disclosed methods forinhibiting the growth of cancer cells and for the treatment of cancers,including ovarian cancers, such as small cell carcinomas of the ovary(SCCO), in particular SCCO hypercalcemic type (SCCOHT), which cancersare associated with a cancer cell exhibiting reduced or undetectableSMARCA4 gene expression and/or with reduced or undetectable SMARCA4protein levels and/or protein function. Exemplified herein arecompounds, including polynucleotides, polypeptides, and small moleculesthat can be used to restore SMARCA4 gene expression and/or functionand/or restore SMARCA4 protein level and/or function and, thereby, slowor stop the growth of a cancer cell; and

4. Diagnostic kits for identifying a cancer cell or a detecting in apatient a cancer cell that exhibits reduced or eliminated SMARCA4 geneexpression and/or function and/or reduced or absent SMARCA4 proteinlevel and/or functionality, which diagnostic kits contain one or moreagents that can be used alone or in combination to: (a) detect amutation in a SMARCA4 gene and/or mRNA, such as an insertion mutation, adeletion mutation, a frame shift mutation, a splice site mutation, and apoint mutation, in particular nonsense mutation and/or a missensemutation in a SMARCA4 gene and/or mRNA; (b) detect a reduction inSMARCA4 mRNA level; (c) detect a reduction in SMARCA4 protein level;and/or (d) detect a reduction in SMARCA4 protein functionality.

These and other aspects of the present disclosure can be betterunderstood by reference to the following non-limiting definitions.

DEFINITIONS

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the presently disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about” meaning approximately. Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand attached claims are approximations that can vary depending upon thedesired properties sought to be obtained by the presently disclosedsubject matter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, the term “gene” is used broadly to refer to any segmentof DNA associated with a biological function. Thus, genes include, butare not limited to, coding sequences and/or the regulatory sequencesrequired for their expression. Genes can also include non-expressed DNAsegments that, for example, form introns or recognition sequences for apolypeptide. Genes can be obtained from a variety of sources, includingcloning from a source of interest or synthesizing from known orpredicted sequence information, and can include sequences designed tohave desired parameters.

As used herein, the terms “peptide,” “protein” and “polypeptide” referto any polymer comprising any of the 20 protein amino acids, regardlessof its size. Although “protein” is often used in reference to relativelylarge polypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and varies. Theterm “protein” as used herein refers to peptides, polypeptides andproteins, unless otherwise noted. As used herein, the terms “protein”,“polypeptide” and “peptide” are used interchangeably herein whenreferring to a gene expression product.

As used herein, the term “polynucleotide” refers to a nucleic acidmolecule, such as mRNA, RNA, cRNA, cDNA or DNA. The term typicallyrefers to oligonucleotides greater than 30 nucleotide residues inlength. Smaller nucleic acid molecules, generally between 5 and 30nucleotides long, are referred to as oligonucleotides. ***

As used herein, the term “SWI/SNF related, matrix associated, actindependent regulator of chromatin, subfamily a, member 4” or “SMARCA4”refers to a protein that in humans is encoded by the SMARCA4 gene, whichis located on the short (p) arm of chromosome 19 at position 13.2.SMARCA4 is also known as, ATP-dependent helicase SMARCA4, BAF190,BAF190A, brahma protein-like 1, BRG1, BRG1-associated factor 190A,BRM/SWI2-related gene 1, FLJ39786, hSNF2b, MRD16, nuclear protein GRB1,protein brahma homolog 1, protein BRG-1, RTPS2, SMCA4_HUMAN, SNF2,SNF2-beta, SNF2L4, SNF2LB, SNF2-like 4, sucrose nonfermenting-like 4,SWI2, or transcription activator BRG1. The SMARCA4 protein forms asubunit of several different SWI/SNF protein complexes. SWI/SNFcomplexes regulate gene expression by a process known as chromatinremodeling. Chromatin is the network of DNA and protein that packagesDNA into chromosomes. The structure of chromatin can be changed(remodeled) to alter how tightly DNA is packaged. Chromatin remodelingis one way gene expression is regulated during development; when DNA istightly packed, gene expression is lower than when DNA is looselypacked. Exemplary sequences of SMARCA4 mRNA, genomic DNA and protein areprovided in SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7 and 8.

As used herein, the term “SWI/SNF complex” refers to a protein complexthat includes SMARCA4 and isoforms of ARID1, SMARCC, SMARCD, ACTL6proteins. In eukaryotes, the SWI/SNF complex plays an important role innucleosome remodeling. SWI/SNF complex (in yeast) is capable of alteringthe position of nucleosomes along DNA. Whitehouse et al., Nature400(6746): 784-7 (1999). Through their ability to regulate geneactivity, SWI/SNF complexes are involved in many processes, includingrepairing damaged DNA; copying (replicating) DNA; and controlling thegrowth, division, and maturation (differentiation) of cells. The BRG1protein and other SWI/SNF subunits are thought to act as tumorsuppressors, which keep cells from growing and dividing too rapidly orin an uncontrolled way. It was first identified in 1998 as a tumorsuppressor in rhabdoid tumors, a rare pediatric malignancy. Versteege etal., Nature 394(6689): 203-6 (1998). As DNA sequencing costs diminished,many tumors were sequenced for the first time around 2010. Several ofthese studies revealed SWI/SNF to be a tumor suppressor in a number ofdiverse malignancies. Wiegand et al., N. Engl. J. Med. 363(16): 1532-43(2010).

As used herein, the term “mutation” refers to any modification in thenucleotide sequence of a nucleic acid relative to a wild-type nucleicacid sequence. Mutations include, without limitation, insertionmutations, deletion mutations, frame shift mutations, splice sitemutations, and point mutations, in particular nonsense mutations and/ormissense mutations. Silent mutations, and more generally mutationshaving no deleterious effect on the sequence of an expression product oron its regulation are not contemplated for the products and methods ofthe present disclosure. Of particular interest herein are mutations thatresult in reduced or eliminated expression of SMARCA4 or in reduced oreliminated resulting SMARCA4 protein function (as defined below).

As used herein, the term “polymorphism” refers to the occurrence of twoor more genetically determined alternative variant sequences (i.e.,alleles) occurring in a population. A polymorphic marker is the locus atwhich divergence occurs. Preferred markers have at least two alleles,each occurring at a frequency of greater than 1%. A polymorphic locusmay be as small as one base pair (e.g., a single nucleotide polymorphism(SNP)). Exemplary SNPs are disclosed herein and can be referenced byaccession number (e.g., “rs number”). The rs numbers (searchable throughNCBI's Entrez SNP website) comprise the SNP as well as proximatecontiguous nucleotides provided to place the SNP in context within thegene. Thus, rs numbers referenced herein are intended to indicate thepresence of the SNP and not to require the presence of all or part ofthe contiguous nucleotide sequence disclosed therein. Further, referenceto a particular polymorphism is intended to also encompass thecomplementary nucleotide(s) on the complementary nucleotide strand(e.g., coding and non-coding polynucleotides).

As used herein, the term “reference genotype” as used herein refers to apreviously determined pattern of genetic variation associated with aparticular phenotype, such as for example male infertility due to lowsperm motility and/or impaired mitochondrial function. The referencegenotype can be as minimal as the determination of a single base pair,as in determining one or more polymorphisms in the subject. Further, thereference genotype can comprise one or more haplotypes. Still further,the reference genotype can comprise one or more polymorphisms exhibitinghigh linkage disequilibrium to at least one polymorphism or haplotype.In some particular embodiments, the reference genotype comprises one ormore polymorphisms (e.g., SNPs) and/or haplotypes of SMARCA4, SMARCA2,or combinations thereof determined to be associated with maleinfertility due to low sperm motility and/or impaired mitochondrialfunction. In some embodiments, the haplotypes represent a particularcollection of specific single nucleotide polymorphisms.

As used herein, the terms “plasmid” and “vector” refer to any of a widevariety of nucleic acids into which a desired sequence may be insertedby restriction and ligation for transport between different geneticenvironments or for expression in a host cell. Vectors are typicallycomposed of DNA, although RNA vectors are also available. Vectorsinclude, but are not limited to, plasmids and phagemids. Expressionvectors and cloning vectors are examples of vectors that are able toreplicate in a host cell, and that typically include a region, oftendefined by a multiple cloning site, into which a nucleic acid may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. Expression vectors further include one or more transcriptionalpromoters that facilitate the binding of a polymerase, which permits theexpression of the exogenous gene or cDNA.

As used herein, the term “RNAi expression vector” (also referred toherein as a “dsRNA-encoding plasmid”) refers to replicable nucleic acidconstructs used to express (transcribe) RNA which produces siRNAmoieties in the cell in which the construct is expressed. Such vectorsinclude a transcriptional unit comprising an assembly of (1) geneticelement(s) having a regulatory role in gene expression, for example,promoters, operators, or enhancers, operatively linked to (2) a “coding”sequence which is transcribed to produce a double-stranded RNA (two RNAmoieties that anneal in the cell to form an siRNA, or a single hairpinRNA which can be processed to an siRNA), and (3) appropriatetranscription initiation and termination sequences. The choice ofpromoter and other regulatory elements generally varies according to theintended host cell.

As used herein, the term “expression” generally refers to the cellularprocesses by which an RNA is produced by RNA polymerase (RNA expression)or a polypeptide is produced from RNA (protein expression). Thus theterm “expression” describes levels of either RNA or protein in a cellthat can be quantified by methods described in the disclosure.

As used herein, the terms “loss-of-function” or “dysfunctional” or “lossof expression” variously refer to a reduced level of expression of agene (either in RNA or in protein) or reduced or absent proteinproduction or to a decreased ability of a gene to perform its biologicalfunction, e.g. to bind to another protein such as a receptor, to bind toDNA in one cell when compared to the level in another cell, or in onecondition when compared to another condition. As used herein,“loss-of-function,” “dysfunctional,” or “loss of expression” refers to areduction in gene expression, protein production, or protein activitythat is from about 0% to about 50%, or from about 0% to about 40%, orfrom about 0% to about 30%, or from about 0% to about 20%, or from 0% toabout 10%, or less than about 5% of that observed in a cell exhibitingnormal or wild-type gene expression, protein production, or proteinactivity with respect to SMARCA4. SMARCA4 functions include both directand downstream functions as described below.

As used herein, the term “gain-of-function” refers to an increase in thelevel of expression of a gene (either in RNA or in protein) or to anenhanced ability to perform its direct or indirect biological function,e.g. to bind to another protein such as a receptor, to bind to DNA inone cell when compared to the level in another cell, or in one conditionwhen compared to another condition. As used herein, “gain-of-function”refers to an increase in gene expression, protein production, or proteinactivity in a cell that is from about 2-fold to about 1000-fold, or fromabout 5-fold to about 500-fold, or from about 10-fold to about 200-fold,or from about 20-fold to about 100-fold, or from about 30-fold to about70-fold higher in a cell exhibiting normal or wild-type gene expression,protein production, or protein activity than in a cell exhibiting “aloss of function,” a “dysfunction,” or a loss of expression of said geneor its ability to perform its biological function.

As used herein, the term “restoration” refers to an increase inexpression of a previously deficient gene (either in RNA or in protein)or to an enhanced ability of such a gene to perform its biologicalfunction, e.g. to bind to another protein such as a receptor, to bind toDNA, in a cancer cell or in a cell that is otherwise exhibitingdysfunction or deficiency of this gene, to a level comparable to, equalto or higher than the expression or ability of the same gene in anon-cancer normal cell.

As used herein, “significance” or “significant” relates to a statisticalanalysis of the probability that there is a non-random associationbetween two or more entities. To determine whether or not a relationshipis “significant” or has “significance”, statistical manipulations of thedata can be performed to calculate a probability, expressed as a“p-value”. Those p-values that fall below a user-defined cutoff pointare regarded as significant. A p-value in some embodiments less than orequal to 0.05, in some embodiments less than 0.01, in some embodimentsless than 0.005, and in some embodiments less than 0.001, are regardedas significant.

As used herein, the term “biological sample” refers to a tissue or fluidsample obtained from a patient, typically such a biological sample is atumor sample, including an ovarian tumor sample, in particular an SCCOHTsample such as a tissue or biopsy sample (e.g., tumor biopsy).Biological samples may also include sections of tissues such as frozensections taken for histological purposes.

As used herein, the term “diagnosed” refers to a determination that hasbeen made that the cancer is, for example, a SCCOHT, an ovarian cancer,a lung cancer, or other cancer that exhibits dysfunctional and/ordeficient SMARCA4. A diagnosis may be made prior to (on a differentsample) performing the present methods for inhibiting the growth and/orsurvival of a SCCOHT, an ovarian cancer, a lung cancer, or other cancerthat exhibits dysfunctional and/or deficient SMARCA4 or a diagnosis maybe made in conjunction (i.e., either concurrently or sequentially) withthe present methods for inhibiting the growth and/or survival of aSCCOHT, an ovarian cancer, a lung cancer, or other cancer that exhibitsdysfunctional and/or deficient SMARCA4.

The term “reduced SMARCA4 protein function” is meant to includereduction in a direct or indirect (downstream) biological functions. Asused herein, the term “SMARCA4 protein function” refers to any of thedirect or indirect (downstream) activities of SMARCA4. An example of adirect activity is its participation in the SWI/SNF complex. An exampleof a downstream activity includes any of the activities described abovefor the SWI/SNF complex, such as the tumor suppressing activity ofSMARCA4 as well as any pathways by which such downstream activities aremediated, for example inhibition of EZH2 hyperactivity as describerfurther herein. Reductions in function can be brought about for exampleby an altered SMARCA4 protein structural configuration or conformation,an altered SMARCA4 protein post-translational modification, an alteredlevel of protein-protein complex formation between a SMARCA4 protein andanother protein, such as altered SWI/SNF or BAF complex formation and/oran altered level of one or more SMARCA4 downstream target genes, orcorresponding mRNA, such as one or more of the CDH1, CDH3, EHF, RRAD,and/or ML-IAP genes or mRNA. As used herein, reduced SMARCA4 proteinfunction includes a reduction in SMARCA4 protein function that is fromabout 0% to about 50%, or from about 0% to about 40%, or from about 0%to about 30%, or from about 0% to about 20%, or from 0% to about 10%, orless than about 5% of the protein functionality in a cell exhibitingnormal or wild-type SMARCA4 protein function

As used herein, the term “identifying” refers to an initialdetermination, such as a determination that a cancer cell exhibitsdysfunctional and/or deficient SMARCA4 and/or that a cancer cellexhibits enhanced susceptibility to a therapeutic compound that enhancesSMARCA4 functionality and/or promote SMARCA4 expression. “Identifying”does not determine the selection of a final medical treatment regimen,but may be used by a skilled clinician in designing and/or selectingsuch a treatment regimen.

As used herein, the terms “treatment” and “treating” refer totherapeutic regimen that inhibits (reduces or arrests or causesapoptosis) the growth of a cancer cell in patient by administering tothe patient an compound that increases and/or restores SMARCA4 geneexpression and/or protein functionality and/or by administering to thepatient a wild-type SMARCA4 gene or a wild-type SMARCA4 protein.Suitable treatments include the administration to the patient of aSMARCA4 gene or polynucleotide sequence that increases SMARCA4 geneexpression that is a component of (a) a viral vector, such as aretroviral vector, an adenoviral vector, or a vaccinia-viral vector, (b)a liposome, or (c) a nano-particle. Suitable treatments also include theadministration to the patient of a SMARCA4 protein that is a componentof a liposome or a nanoparticle. Alternative suitable treatments includethe administration to the patient of an antibody, shRNA, siRNA, orantisense-oligonucleotide that reduces SMARCA2 gene expression.

As used herein, the terms “small cell carcinoma of the ovary,hypercalcemic type” and “SCCOHT” refer to a rare and aggressive form ofovarian cancer. SCCOHT is still categorized as a miscellaneous tumor bythe World Health Organization. Most tumors are unilateral, and sizegreater than 10 cm may be prognostically favorable due to earlier onsetof symptoms resulting in stage migration. Estel et al., Arch GynecolObstet 284:1277-82 (2011). Histologic classification can be challenging,but commonly expressed immunohistochemical markers such as CD10, WT1,and calretinin can be useful in the setting of absent inhibin, S100, andchromogranin expression to exclude histological mimics. McCluggage, AdvAnat Pathol 11:288-96 (2004).

An “allele” refers to any of two or more alternative forms of a genethat occupy the same locus on a chromosome. If two copies of same alleleare present in an individual, the individual is homozygous for thatallelic form of the gene. If different alleles are present in anindividual, the individual is heterozygous for that gene.

The term “primer” refers to a nucleic acid capable of acting as a pointof initiation of synthesis along a complementary strand when conditionsare suitable for synthesis of a primer extension product. Thesynthesizing conditions include the presence of four different bases andat least one polymerization-inducing agent such as reverse transcriptaseor DNA polymerase. These are present in a suitable buffer, which mayinclude constituents which are co-factors or which affect conditionssuch as pH and the like at various suitable temperatures. A primer ispreferably a single strand sequence, such that amplification efficiencyis optimized, but double stranded sequences can be utilized.

The term “probe” refers to a nucleic acid that hybridizes to a targetsequence. In some embodiments, a probe includes about eight nucleotides,about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about25 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50nucleotides, about 60 nucleotides, about 70 nucleotides, about 75nucleotides, about 80 nucleotides, about 90 nucleotides, about 100nucleotides, about 110 nucleotides, about 115 nucleotides, about 120nucleotides, about 130 nucleotides, about 140 nucleotides, about 150nucleotides, about 175 nucleotides, about 187 nucleotides, about 200nucleotides, about 225 nucleotides, and about 250 nucleotides. A probecan further include a detectable label. A detectable label can becovalently attached directly to a probe oligonucleotide, e.g., locatedat the probe's 5′ end or at the probe's 3′ end.

The terms “hybridize” or “hybridization,” as is known to those ofordinary skill in the art, refer to the binding or duplexing of anucleic acid molecule to a particular nucleotide sequence under suitableconditions, e.g., under stringent conditions.

As used herein, the term “genotype” means a sequence of nucleotidepair(s) found at one or more sites in a locus on a pair of homologouschromosomes in an individual. Genotype may refer to the specificsequence of the gene.

Methods for the Detection of Small Cell Carcinoma of the Ovary

The present disclosure provides methods for the diagnosis of ovariancancers, such as small cell carcinomas of the ovary (SCCO), inparticular SCCO hypercalcemic type (SCCOHT), which cancers areassociated with reduced or undetectable SMARCA4 gene expression and/orwith reduced or undetectable SMARCA4 protein levels and/or proteinfunction, which methods include the detection of one or more mutations,including one or more germline or somatic mutations, in the SMARCA4 geneand/or associated regulatory sequences in particular mutations in bothalleles of the SMARCA4 gene and/or associated regulatory sequences(i.e., bi-allelic mutations), which are known, predicted, ordemonstrated to reduce or eliminate SMARCA4 gene expression and/orknown, predicted, or demonstrated to reduce or eliminate SMARCA4 proteinlevels and/or protein function.

As disclosed herein, it was discovered that mutations in the SMARCA4gene are highly distinctive markers for SCCOHT cancer cells and patientsafflicted with SCCOHT or in any event having SCCOHT cancer cells. Amongall 12 SCCOHT patient samples characterized, sequencing of allprotein-coding exons in 279 cancer-related genes for 12 paired tumor andnormal SCCOHT samples identified SMARCA4 gene mutations, typicallybi-allelic SMARCA4 gene mutation (FIG. 1B), that resulted in reducedSMARCA4 gene expression and/or functionality. Moreover, it wasdetermined that the probability of identifying SMARCA4 mutations in all12 samples was less than 2.22×10⁻¹⁶.

Mutations, including loss-of-function/loss-of-expression mutations,within the SMARCA4 gene were identified in each of the 12 SCCOHTpatients studied (see, Table 1), which cause the SMARCA4 gene to eitherencode reduced or undetectable levels of SMARCA4 mRNA, to encode SMARCA4mRNA that cannot be transcribed into SMARCA4 protein, and/or to encodemRNA that does not encode functional SMARCA4 protein. Examples of suchmutations include insertion mutations, deletion mutations, frame shiftmutations, splice site mutations, nonsense mutations, and missensemutations, which result in reduced or undetectable levels of SMARCA4protein or SMARCA4 protein having reduced or undetectable functionality.

In the 12 SCCOHT patients studied, several of SMARCA4 gene mutationswere non-sense or frameshift mutations that occurred within or upstreamof the region of the SMARCA4 gene encoding the SMARCA4 helicase and SNF2functional domains. Because the helicase and SNF2 functional domains areessential for SMARCA4 functionality, those mutations inactivated theSMARCA4 protein, which was frequently undetectable byimmunohistochemistry analysis (IHC).

Thus, as disclosed herein, the detection of mutations within the SMARCA4gene or mRNA transcript that are predicted to result in reduced orundetectable intracellular levels of SMARCA4 protein or that arepredicted to result in SMARCA4 protein having reduced or undetectablefunctional activity can be exploited diagnostically to determine if acancer cell exhibits the SCCOHT phenotype, or conversely, to excludeSCCOHT.

As further disclosed herein, the determination that a SMARCA4 proteinexhibits reduced or undetectable functionality or the detection oftruncated, or otherwise structurally altered SMARCA4 protein, are allsimilarly predictive of SCCOHT and, therefore can also be exploiteddiagnostically to determine if a cancer cell exhibits the SCCOHTphenotype or to exclude same.

Methods and Kits

The present disclosure provides reagents, methods, and kits fordetermining the presence and/or amount of: a) at least one loss offunction mutation in a SMARCA4 gene; b) mutant mRNA encoding SMARCA4protein; and/or c) mutant SMARCA4 protein in or isolated from abiological sample.

Methods include a method of detecting the presence of a loss of functionmutation in a SMARCA4 nucleic acid sequence, comprising: isolating anucleic acid that comprises a nucleic acid that encodes a portion of aSMARCA4 protein or that comprises a portion of the SMARCA4 gene, whereinthe nucleic acid comprises a nucleotide position that can be mutated ascompared to a reference sequence, wherein when the nucleotide positionis mutated a function of SMARCA4 protein is decreased or eliminated, andsequencing the isolated nucleic acid to determine whether the nucleotidein the nucleotide position is mutated as compared to the referencesequence. Another method provides a method of detecting the presence ofa loss of function mutation in a SMARCA4 nucleic acid sequence,comprising: contacting the nucleic acid that comprises a nucleic acidthat encodes a portion of a SMARCA4 protein or that comprises a portionof the SMARCA4 gene with a primer or probe under conditions suitable forhybridization and/or amplification, wherein the nucleic acid comprises anucleotide position that can be mutated as compared to a referencesequence, wherein when the nucleotide position is mutated a function ofSMARCA4 protein is eliminated or decreased, and determining whether thenucleic acids hybridize to one another and/or determining the sizeand/or sequence of the amplified region.

In other embodiments, a method for determining whether the nucleic acidshybridize to one another comprises determining whether a mismatch ispresent by contacting the hybridized sample with an agent that cleavesat the site of a mismatch, and identifying the size of any of theproducts of the cleavage reaction, wherein if a mismatch is present acleavage product is detected.

In some embodiments, the method involves detecting a germline mutationusing an array or probe designed to distinguish mutations in a SMARCA4gene. Mutations include insertions, deletions, and substitutions. Insome embodiments, substitutions result in the formation of stop codons.In other embodiments, insertions or deletions result in frameshift ormissense mutations. Probes or cDNA oligonucleotides that detectmutations in a nucleic acid sequence can be designed using methods knownto those of skill in the art.

In some embodiments, mutations are identified as those that lead to adecrease in expression of SMARCA4 or in expression of a SMARCA4 proteinwith decreased or eliminated function. In some embodiments, the mutationis a missense, frameshift, or stop codon mutation. In an embodiment, themutation results in a carboxy-terminal truncation of the SMARCA4protein. In some embodiments, the mutations are one or more or all themutations shown in Table 1.

In some embodiments, the methods and kits may provide restrictionenzymes and/or probes that can detect changes to the restrictionfragments as a result of the presence of at least one mutation in thegene sequence encoding SMARCA4 protein. The publicly available humangenome sequence can be used for example to generate a RFLP map.

In some embodiments, the methods and kits provide for primers and probesthat can detect the presence of at least one mutation in the mRNA and/ordetect an alteration in size or sequence of mRNA. In some embodiments,primers are designed to hybridize within a certain temperature range andmay also include other sequences such as universal sequencing sequences.In some embodiments, the target sequence of the primer/probe setsincludes those that are complementary to mature coding sequence. Thoseprimer/probes can act as a positive control to detect full lengthtranscripts that encode active SMARCA4 protein. In some embodiments, theprimers and probes complementary to the 3′ untranslated region areexcluded as positive controls in order to avoid spurious detection ofdegraded mRNA and to enhance the correlation between the mRNA that ismeasured by this assay and the protein that is actually expressed.

In some embodiments, the kit can include one or more probes and/orprimer attached to a solid substrate. In some embodiments, an array cancomprise one or more of SMARCA4 gene-specific sequences. In someembodiments, the array or kit excludes detection of a gene selected fromthe group consisting of actin, gapdh, aldolase, hexokinase, cyclophilinand combinations thereof.

In some embodiments, the methods and kits provide reagents for detectionof the presence or absence of the SMARCA4 protein. In some embodiments,the reagents include an antibody that can detect full length SMARCA4protein in cells. In other embodiments, an antibody can detectpolypeptides that have an alteration in one or more domains of theSMARCA4 polypeptide. The antibodies can be detectably labeled.Detectable labels include for example fluorescent labels, radioactiveisotope labels, and polypeptide labels including enzymes or moleculeslike biotin. The methods of detection involve for exampleimmunohistochemical or radiological detection of SMARCA4 protein oraltered SMARCA4 protein in tumor tissue.

The kit can establish patterns of SMARCA4 expression that may beassociated with certain cancers, including ovarian cancers, inparticular SCCOHT. The presence of a SMARCA4 mutation can be used toprognosticate risk of malignancy, diagnose or confirm or excludediagnosis of SCCOHT in a patient, and help identify appropriatetreatment.

The disclosure provides a method of determining the diagnosis orprognosis of ovarian cancer comprising: determining whether the gene ornucleic acid that comprises a nucleic acid that encodes a portion of aSMARCA4 protein or that comprises a portion of the SMARCA4 gene has thereference sequence or the mutated sequence. In some embodiments, theexpression or decrease in expression in a cell or tissue sample can bedetermined for example by PCR analysis, hybridization analysis, in situanalysis using hybridization, or antibody detection methods.

In some embodiments, the cancer is selected from the group consisting ofovarian cancer, lung cancer, or other cancer that exhibits dysfunctionaland/or deficient SMARCA4 as described herein.

In some embodiments, once a cancer is diagnosed in a patient otherfamily members may also be examined for the presence or absence of aloss of function mutation in SMARCA4.

In some embodiments, after detection of one or more mutations in SMARCA4is detected, a treatment is selected and administered to the patient. Amethod of treating a cancer, comprising administering to a cancerpatient, one or more compounds, including one or more polynucleotides,polypeptides, and/or small molecules that can restore SMARCA4 geneexpression and/or SMARCA4 protein levels and/or protein function and,thereby, slow or stop the growth of the cancer cell.

Each of these aspects of the present disclosure is described in furtherdetail in the following sections.

1. Methodology for Detecting Mutations in a SCCOHT Nucleotide Sequence

In one aspect, the present disclosure provides methods for identifying acancer cell or a patient with a cancer cell that exhibits reduced orundetectable SMARCA4 protein functionality or reduced or undetectableSMARCA4 protein levels, wherein the methods include detecting a mutationin a SMARCA4 gene or a SMARCA4 mRNA, such as a nonsense mutation, amissense mutation, a deletion mutation, an insertion mutation, aframeshift mutation, and/or a splice site mutation in a SMARCA4 gene ora SMARCA4 mRNA, which mutation is predictive of reduced SMARCA4 proteinlevels and/or functionality.

The detection of mutations in a SMARCA4 gene or a SMARCA4 mRNA,including the detection of nonsense mutations, missense mutations,deletion mutations, insertion mutations, frameshift mutations, and/orsplice site mutations in a SMARCA4 gene or a SMARCA4 mRNA can beachieved by routine modification of methodologies that are readyavailable to and known by those of skill in the art in conjunction withthe reported nucleotide sequences for the SMARCA4 gene and/or a SMARCA4mRNA, which are presented herein as SEQ ID NOs 1, 3, 5, and 7. Exemplarymethodologies for detecting mutations in a nucleotide sequence (e.g.,genomic DNA, mRNA, cDNA) include amplification, sequencing, andhybridization methodologies, some of which are presented herein toexemplify rather than limit the presently-disclosed methods fordetection of SCCOHT, which can be applied by routine modification toother cancers associated with mutations in the SMARCA4 gene.

(a) SMARCA4 Functional Domains and Activities

The following summarizes representative activities of SMARCA4, includingthe role of those activities in the formation of protein-proteincomplexes as well as the structural basis for those activities (Reismanet al., Oncogene 28:1653-68 (2009) and Wilson and Roberts, Nat RevCancer 11:481-92 (2011)). Based upon this description of thoseactivities, one skilled in the art will readily recognize the nature ofmutations, insertions, and/or substitutions within the gene encodingSMARCA4 protein that will reduce and/or eliminate one or more of thoseactivities and, therefore, the functionality of the SMARCA4 protein.

The SMARCA4 protein contains the following conserved domains: (1) aproline rich region, containing more than 25% of proline residues in theamino acid sequence; (2) HSA and BRK domains, containing motifs that maypredict binding to DNA; (3) an ATPase/helicase domain, contains motifspresent in the DEAD helicases superfamily; and (4) a bromodomain, foundin many chromatin-associated proteins. Singh et al., Biol Chem387(10-11):1469-78 (2006). SMARCA4 is the catalytic subunit of thechromatin-remodeling complex SWI-SNF and influences transcriptionalregulation by disrupting histone-DNA contacts in an ATP-dependentmanner.

SMARCA4 is a key component of the SWI/SNF complex, which functions as amaster regulator of gene expression through chromatin remodeling toalter nucleosome conformation, making it more accessible totranscriptional activation. Reisman et al., Oncogene 28:1653-68 (2009)and Wilson and Roberts, Nat Rev Cancer 11:481-92 (2011). SMARCA4'simportance has also been demonstrated in its function to regulate theexpression of genes known to be involved in tumor suppression, apoptosisand epigenetics such as cadherin 1 (CDH1), cadherin 3 (CDH3), ETShomologous factor (EHF), Ras-related associated with diabetes (RRAD) andmelanoma inhibitor of apoptosis (ML-IAP). Song et al., Mol Cancer Res0427 (2013), Published Online Jan. 20, 2014; Saladi et al., Pigment CellMelanoma Res. 26(3): 377-91 (2013).

(b) Amplification

Detection of SMARCA4 mutation may be performed by nucleic acidamplification. As used herein, the term “amplification” refers to theproduction of multiple copies of a target nucleic acid that contains atleast a portion of the intended specific target nucleic acid sequence.The multiple copies are referred to, interchangeably, as amplicons oramplification products. In certain aspects of the present disclosure,the amplified target contains less than the complete target mRNAsequence (i.e., spliced transcript of exons and flanking untranslatedsequences) and/or target genomic sequence (including introns and/orexons). For example, specific amplicons may be produced by amplifying aportion of the target polynucleotide by using amplification primers thathybridize to, and initiate polymerization from, internal positions ofthe target polynucleotide. The amplified portion contains a detectabletarget sequence that may be detected using any of a variety ofwell-known methods.

The polymerase chain reaction (PCR; described for example in U.S. Pat.Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188) uses multiple cycles ofdenaturation, annealing of primer pairs to opposite strands, and primerextension to exponentially increase copy numbers of the target sequence.In a variation called RT-PCR, reverse transcriptase (RT) is used to makea complementary DNA (cDNA) from mRNA, and the cDNA is then amplified byPCR to produce multiple copies of DNA.

Total RNA may be extracted from cancer tissue samples or cells andnon-cancer control tissue samples or cells using Trizol recompound.First strand synthesis may be carried out using 1-2 μg of total RNA withSuperScript II reverse transcriptase (Life Technologies, Carlsbad,Calif.) at 42° C. for one hour. cDNA may then be amplified by PCR withSMARCA4 gene-specific primers that are designed based upon SMARCA4nucleotide sequences presented in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, and SEQ ID NO: 7, or that are otherwise known and readily availableto those skilled in the art. Specific examples of useful DNA polymeraseinclude LA Taq DNA polymerase (Takara), Ex Taq polymerase (Takara), GoldTaq polymerase (Perkin Elmer), AmpliTaq (Perkin Elmer), Pfu DNApolymerase (Stratagene) and the like.

Subsequently, the amplified product can be subjected to agarose gelelectrophoresis, followed by staining with ethidium bromide, SYBR Greensolution or the like to thereby detect the amplified product as a bandor two to three bands (DNA fragments). Thus, a part of a gene encodingSMARCA4, containing a genetic polymorphism can be detected as a DNAfragment. Instead of agarose gel electrophoresis, polyacrylamide gelelectrophoresis or capillary electrophoresis may be performed. It isalso possible to perform PCR using primers labeled in advance with asubstance such as fluorescent dye and to detect the amplified product. Adetection method which does not require electrophoresis may also beemployed; in such a method, the amplified product is bound to a solidsupport such as a microplate, and a DNA fragment of interest is detectedby means of fluorescence, enzyme reaction, or the like.

For example, TaqMan PCR can be used to detect SMARCA4 gene deletions orinsertions. TaqMan PCR is a method using PCR reaction with fluorescentlylabeled allele-specific oligos and Taq DNA polymerase. Theallele-specific oligo used in TaqMan PCR (called “TaqMan probe”) may bedesigned based on the SNP information and need not amplify the wholegene or even a whole exon or intron. The 5′ end of TaqMan probe islabeled with fluorescence reporter dye R (e.g. FAM or VIC), and at thesame time, the 3′ end thereof is labeled with quencher Q (quenchingsubstance). Thus, under these conditions, fluorescence is not detectablesince the quencher absorbs fluorescence energy. Since the 3′ end ofTaqMan probe is phosphorylated, no extension reaction occurs from TaqManprobe during PCR reaction. However, when PCR reaction is performed usingthis TaqMan probe together with Taq DNA polymerase and primers designedso that an SNP-containing region is amplified, the reaction describedbelow occurs.

First, a TaqMan probe hybridizes to a specific sequence in the templateDNA, and at the same time, an extension reaction occurs from a PCRprimer. At this time, Taq DNA polymerase having 5′ nuclease activitycleaves the hybridized TaqMan probe as the extension reaction of PCRprimer proceeds. When the TaqMan probe has been cleaved, the fluorescentdye becomes free from the influence of the quencher. Then, fluorescencecan be detected.

For example, two alleles are supposed: one allele has A at the SNP site(allele 1) and the other allele has G at the SNP site (allele T). ATaqMan probe specific to allele 1 is labeled with FAM and another TaqManprobe specific to allele 2 is labeled with VIC. These two allelespecific oligos are added to PCR recompounds, and then TaqMan PCR isperformed with a template DNA whose SNP is to be detected.

Subsequently, fluorescence intensities of FAM and VIC are determinedwith a fluorescence detector. When the SNP site of the allele iscomplementary to the site within TaqMan probe corresponding to the SNP,the probe hybridizes to the allele; and Taq polymerase cleaves thefluorescent dye of the probe, which becomes free form the influence ofthe quencher. As a result, fluorescence intensity is detected.

Other PCR amplification methods can also be used. Any suitable PCRmethod for detecting SNPs in target DNA are contemplated by the presentinvention, including new methods, such as the invader PCR method (e.g.,Allawi et al, J Clin Aicrobio 44: 3443-47 (2006), incorporated herein byreference) or the SniPer PCR method (Huentelman et al., BMC Genomics6:149 (2005), incorporated herein by reference), both of which can beused to detect SNPs.

In addition to detect point mutations, PCR amplification methods canalso be used together with other common methods to detect deletions,insertions of the SMARCA4 genes. For example, amplified SMARCA4 orportions thereof can be applied to an agarose gel and the length of theSMARCA4 PCR products can be deduced by comparing to the known length ofwide-type SMARCA4 genomic sequence or cDNA, or corresponding portionsthereof. Primers can be selected to amplify targeted portions of theSMARCA gene and can be labelled for detection. Primers can be includedfor co-amplification of positive and negative controls.

(c) Sequencing

Mutations of SMARCA4 gene can be determined by the direct sequencing ofgenomic DNA, cDNA or mRNA in a cancer patient tissue sample or celland/or a non-cancer donor control tissue sample or cell. Alternatively,mutation of SMARCA4 gene can be determined following conversion of mRNAinto cDNA by reverse transcription. Multiple methods can be used tosequence SMARCA4 nucleotide sequence. After SMARCA4 is sequenced,loss-of-function mutations can be detected by examining the locations ofthe mutations, the effects of the mutations on critical functionaldomains of SMARCA4, and other bioinformatics analysis methods.

Nucleotide sequencing can be achieved through chain termination methods,which were first developed by Frederick Sanger, and can be referred toas Sanger sequencing methods. In chain termination methods, four PCRreactions are performed wherein each reaction is spiked with a singledideoxynucleotide (ddNTP), which is a nucleotide lacking a 3′ hydroxylgroup (e.g., ddATP, ddTTP, ddCTP, ddGTP). When a ddNTP is incorporatedinto a nascent chain of DNA, synthesis of the nascent chain is halted;this generates a mixture of variable length oligonucleotides that can beresolved by size using, for example, DNA electrophoresis in a slab gelor capillary. Any number of detection methods can be used to read theDNA sequence as determined by the relative lengths of oligonucleotidesin each of the four reactions, for example, autoradiography, UV lightdetection, or fluorescent dye detection. Dye termination methods are avariation of chain termination methods whereby each type of ddNTP (e.g.,ddATP, ddTTP, ddCTP, ddGTP) is labeled with a different colorfluorescent dye. This enables DNA to be sequenced in a single PCRreaction.

Direct RNA sequencing technology (Helicos BioSciences Corporation,Cambridge, Mass.) and transcriptome profiling using single-moleculedirect RNA sequencing are described in Ozsolak et al., Nature461(7265):814-818 (2009) and Ozsolak and Milos, Methods Mol Biol 7:51-61(2011). True Single Molecule and Direct RNA Sequencing technologies arefurther described in U.S. Patent Publication Nos. 2008/0081330,2009/0163366, 2008/0213770, 2010/0184045, 2010/0173363, 2010/0227321,2008/0213770, and 2008/0103058 as well as U.S. Pat. Nos. 7,666,593;7,767,400; 7,501,245; and 7,593,109, each of which is herebyincorporated by reference in its entirety.

mRNAs encoded by SMARCA4 gene can be directly sequenced by True SingleMolecule and Direct RNA Sequencing technologies by utilizing specificsequencing primers that are designed based upon the SMARCA4 nucleotidesequences (e.g., as presented in SEQ ID NO's. 1, 3, 5, and 7, or whichare otherwise known and readily available to those skilled in the art).

(d) Hybridization

Hybridization is another method for detecting mutations. This methodinvolving a gene-chip or microarray customized for the SMARCA4 gene.This is particularly useful for detecting reported SMARCA4 mutations.Preferably where a chip or array comprises probes for detection of agenetic variation in SMARCA4, the chip or array comprises one or more ofthe probes as suitable for detection of that genetic variation.

In general the chip or array comprises a support or surface with anordered array of binding (e.g. hybridization) sites or probes. Chip orarray is in general prepared by selecting probes which comprise a givenpolynucleotide sequence, and then immobilizing such probes to a solidsupport or surface. Probes may be designed, tested and selected asdescribed herein. In general the probes may comprise DNA sequences. Insome embodiments the probes may comprise RNA sequences, or copolymersequences of DNA and RNA. The polynucleotide sequences of the probes mayalso comprise DNA and/or RNA analogues, or combinations thereof. Forexample, the polynucleotide sequences of the probes may be full orpartial fragments of genomic DNA. The polynucleotide sequences of theprobes may also be synthesized nucleotide sequences, such as syntheticoligonucleotide sequences. The probe sequences can be synthesized eitherenzymatically in vivo, enzymatically in vitro (e.g., by PCR), ornon-enzymatically in vitro.

A nucleic acid sample, e.g. amplification or fragmentation products,comprising the genetic variation(s) to be detected (target DNA) iscontacted with a probe array as described herein, under conditions whichallow hybridization to occur between target DNA and the correspondingprobes. Specific hybridization complexes are thus formed between targetnucleic acid and corresponding probes.

The hybridization of e.g. fragmentation products, with probes capable ofdetecting corresponding genetic variations deposited on a support may becarried out using conventional methods and devices. In one instance,hybridization is carried out using an automated hybridization station.For hybridization to occur, the e.g. fragmentation products, are placedin contact with the probes under conditions which allow hybridization totake place. Using stable hybridization conditions allows the length andsequence of the probes to be optimized in order to maximize thediscrimination between genetic variations A and B, e.g. between wildtype and mutant sequences, as described herein.

In some embodiments, the method relies on differential hybridization, inparticular an increase in hybridization signal. The method involvesformation of specific hybridization complexes between target DNA andcorresponding probes. Thus target DNA bearing the wild type sequencewill hybridize to the probes designed to detect the wild type sequence,whereas target DNA bearing a mutant sequence will hybridize to theprobes designed to detect that mutant sequence. The hybridizationcomplexes are detectably labelled by means described herein (e.g. thetarget DNA is directly labelled, or both target and probe are labelledin such a way that the label is only detectable on hybridization). Bydetecting the intensity of detectable label (if any) at thepredetermined probe positions it is possible to determine the nature ofthe target DNA in the sample. In this instance the probes (also referredto as allele specific oligonucleotides, ASOs) preferably have thevariable nucleotide(s) at the central position, as described herein.

To distinguish between two known alleles that differ by a single base,three oligonucleotides are necessary: Two are allele-specificoligonucleotides (ASOs) that differ from each other only in the single3′ terminal base; the first is complementary to one allele and thesecond is complementary to the second allele. The third oligonucleotideis complementary to the invariable sequence adjacent to the variantbase. Once hybridization (and optionally post-hybridizationamplification) has taken place, the intensity of detectable label ateach probe position (including control probes) can be determined. Theintensity of the signal (the raw intensity value) is a measure ofhybridization at each probe. The intensity of detectable label at eachprobe position (each probe replica) may be determined using any suitablemeans. The means chosen will depend upon the nature of the label. Ingeneral an appropriate device, for example, a scanner, collects theimage of the hybridized and developed DNA-chip. An image is captured andquantified.

Once the target DNA has been hybridized to the chip and the intensity ofdetectable label has been determined at the probe replica positions onthe chip (the raw intensity values), it is necessary to provide a method(model) which can relate the intensity data from the chip to thegenotype of the individual (otherwise known and readily available tothose skilled in the art).

2. Methodology for Detecting Mutations in SMARCA4 Protein

In addition to nucleotide-based detection methods, mutations can also beidentified through changes in the sequence, 3D structure andimmunoreactivity of the SMARCA4 protein. Mutations such as nonsensemutation, frame shift mutation, addition or deletions often result inthe loss or change of protein immunoreactivity to antibodies. Commonmethods for detecting these changes include western blot,immunoprecipitation, immunohistochemical analysis (IHC),immunofluorescent analysis and proteomics such as LC-MS/MS, and can beperformed by those skilled in the art.

(a) Methodologies for Detecting Structural Alterations (i) Anti-SMARCA4Antibodies

Antibodies for detecting SMARCA4 proteins that can be used in themethods of the present disclosure are widely available commerciallyfrom, for example, LifeSpan BioSciences, Inc. (Seattle, Wash.),Antibodies Online.com (Atlanta, Ga.), PTG Labs (Chicago, Ill.), Abcam®(Cambridge, Mass.), and Santa Cruz Biotechnology (Dallas, Tex.).

Suitable antibodies can also be prepared by using standard techniquesthat are well known and readily available in the art. To preparepolyclonal antibodies or “antisera,” an animal is inoculated with anantigen, i.e., a purified immunogenic SMARCA4 protein, or a peptidethereof. Immunoglobulins are recovered from a fluid, such as bloodserum, that contains the immunoglobulins, after the animal has had animmune response. For inoculation, the antigen is preferably bound to acarrier peptide and emulsified using a biologically suitable emulsifyingcompound, such as Freund's incomplete adjuvant. A variety of mammalianor avian host organisms may be used to prepare polyclonal antibodiesagainst SMARCA4 protein, or a peptide thereof.

Following immunization, immunoglobulin (Ig) can be purified from theimmunized bird or mammal, e.g., goat, rabbit, mouse, rat, or donkey andthe like. For certain applications, particularly certain pharmaceuticalapplications, it is preferable to obtain a composition in which theantibodies are essentially free of antibodies that do not react with theimmunogen. This composition is composed virtually entirely of the hightiter, monospecific, purified polyclonal antibodies to the immunogen.

Antibodies can be purified by affinity chromatography, using purifiedSMARCA4, or a peptide thereof. Purification of antibodies by affinitychromatography is generally known to those skilled in the art (see, forexample, U.S. Pat. No. 4,533,630). Briefly, the purified antibody iscontacted with the purified immunogen bound to a solid support for asufficient time and under appropriate conditions for the antibody tobind to the immunogen. Such time and conditions are readily determinableby those skilled in the art. The unbound, unreacted antibody is thenremoved, such as by washing. The bound antibody is then recovered fromthe column by eluting the antibodies, so as to yield purified,monospecific polyclonal antibodies.

Monoclonal antibodies can be also prepared, using known hybridoma cellculture techniques. In general, this method involves preparing anantibody-producing fused cell line, e.g., of primary spleen cells fusedwith a compatible continuous line of myeloma cells, and growing thefused cells either in mass culture or in an animal species, such as amurine species, from which the myeloma cell line used was derived or iscompatible. Such antibodies offer many advantages in comparison to thoseproduced by inoculation of animals, as they are highly specific andsensitive and relatively “pure” immunochemically. Immunologically activefragments of the present antibodies are also within the scope of thepresent disclosure, e.g., the F(ab) fragment and scFv antibodies, as arepartially humanized monoclonal antibodies.

Thus, it will be understood by those skilled in the art that thehybridomas herein referred to may be subject to genetic mutation orother changes while still retaining the ability to produce monoclonalantibody of the same desired specificity. The present disclosureencompasses mutants, other derivatives and descendants of thehybridomas.

It will be further understood by those skilled in the art that amonoclonal antibody may be subjected to the techniques of recombinantDNA technology to produce other derivative antibodies, humanized orchimeric molecules, fully human recombinant antibodies or antibodyfragments which retain the specificity of the original monoclonalantibody. Such techniques may involve combining DNA encoding theimmunoglobulin variable region, or the complementarity determiningregions (CDRs), of the monoclonal antibody with DNA coding the constantregions, or constant regions plus framework regions, of a differentimmunoglobulin, for example, to convert a mouse-derived monoclonalantibody into one having largely human immunoglobulin characteristics(see EP 184187A, 2188638A, herein incorporated by reference).

A biological sample, e.g., a physiological sample that comprises cancercells from a patient may be lysed to yield an extract which comprisescellular proteins. Alternatively, intact cells, e.g., a tissue samplesuch as paraffin embedded and/or frozen sections of biopsies, arepermeabilized in a manner that permits macromolecules, e.g., antibodies,to enter the cell. The antibodies are then incubated with cells,including permeabilized cells, e.g., prior to flow cytometry, nuclei orthe protein extract, e.g., in a western blot, so as to form a complex.The presence, amount and location of the complex is then determined ordetected.

(ii) Western Blot

The western blot (a/k/a protein immunoblot) is a widely acceptedanalytical technique used to detect specific proteins in the givensample of tissue homogenate or extract. It uses gel electrophoresis toseparate native proteins by 3-D structure or denatured proteins by thelength of the polypeptide. The proteins are then transferred to amembrane (typically nitrocellulose or PVDF), where they are stained withantibodies specific to the target protein. There are now many recompoundcompanies that specialize in providing snyinofird (both monoclonal andpolyclonal antibodies) against tens of thousands of different proteins.WO 2014085698.

Western blot can also be used to detect SMARCA4 mutations because oftenloss-of-function mutations in SMARCA4 are associated with the loss ofamino acids, with the gain of amino acids, and with the change of aminoacids of SMARCA4 that result in the change of SMARCA4's immunoreactivityto certain antibodies.

Samples can be taken from whole tissue or from cell culture. Solidtissues are first broken down mechanically using a bender larger samplevolumes), using a homogenizer (smaller volumes), or by sonication. Cellsmay also be broken open by one of the above mechanical methods. However,virus or environmental samples can be the source of protein and thuswestern blotting is not restricted to cellular studies only.

Assorted detergents, salts, and buffers may be employed to encouragelysis of cells and to solubilize proteins. Protease and phosphataseinhibitors are often added to prevent the digestion of the sample by itsown enzymes. Tissue preparation is often done at cold temperatures toavoid protein denaturing and degradation.

A combination of biochemical and mechanical techniques—comprisingvarious types of filtration and centrifugation—can be used to separatedifferent cell compartments and organelles.

The proteins of the sample can be separated using gel electrophoresis,using such characteristics as isoelectric point (pI), molecular weight,electric charge, or a combination of these. The nature of the separationdepends on the treatment of the sample and the nature of the gel. Thisis a very useful way to identify a protein.

Many modern embodiments of this technique are available, such as thatdisclosed for example in Silva, J. M., McMahon, M. The Fastest Westernin Town: A Contemporary Twist on the Classic Western Blot Analysis. J.Vis. Exp. (84), e51149, doi:10.3791/51149 (2014).

(iii) Immunoprecipitation

As used herein, the term “immunoprecipitation” refers to a technique forprecipitating a protein antigen out of solution using an antibody thatspecifically binds to that particular protein. This process can be usedto isolate and concentrate a particular protein from a sample containinga multitude of different proteins. Immunoprecipitation requires that theantibody be coupled to a solid substrate at some point in the procedure.

Immunoprecipitation can be used to detect SMARCA4 mutations becauseoften loss-of-function mutations in SMARCA4 are associated with the lossof amino acids, with the gain of amino acids, and with the change ofamino acids of SMARCA4 that result in the change of SMARCA4'simmunoreactivity to certain antibodies.

Individual protein immunoprecipitation involves using an antibody thatis specific for a known protein, e.g., SMARCA4, to isolate thatparticular protein out of a solution containing many different proteins.These solutions will often be in the form of a crude lysate of a tissuesample.

(iv) Immunohistochemical Detection

As used herein, the terms “immunohistochemistry” or “IHC” refer to thedetection of SMARCA4 protein in a cell or a tissue section by exploitingthe specific binding of a SMARCA4 antibody to corresponding protein in acancer cell or tissue. Immunohistochemical staining is widely used inthe diagnosis of abnormal cells such as those found in cancerous tumorsand to determine the distribution and localization of biomarkers anddifferentially expressed proteins in different parts of a biologicaltissue. Immunohistochemical detection is described for example in Am JPhysiol Regul Integr Comp Physiol. 2011 September; 301(3): R632-R640.

Visualizing an antibody-antigen interaction can be accomplished in anumber of ways. In the most common instance, an antibody is conjugatedto an enzyme, such as peroxidase, that can catalyze a color-producingreaction. Alternatively, the antibody can also be tagged to afluorophore, such as fluorescein or rhodamine.

The antibodies used for specific detection of SMARCA4 can be polyclonalor monoclonal. Polyclonal antibodies are made by injecting animals withpeptide Ag and, after a secondary immune response is stimulated,isolating antibodies from whole serum. Thus, polyclonal antibodies are aheterogeneous mix of antibodies that recognize several epitopes.Monoclonal antibodies show specificity for a single epitope and aretherefore considered more specific to the target antigen than polyclonalantibodies.

For IHC detection strategies, antibodies are classified as primary orsecondary recompounds. Primary antibodies are raised against an antigenof interest and are typically unconjugated (unlabeled), while secondaryantibodies are raised against immunoglobulins of the primary antibodyspecies. The secondary antibody is usually conjugated to a linkermolecule, such as biotin, that then recruits reporter molecules, or thesecondary antibody itself is directly bound to the reporter molecule.

Reporter molecules vary based on the nature of the detection method, themost popular being chromogenic and fluorescence detection mediated by anenzyme or a fluorophore, respectively. With chromogenic reporters, anenzyme label is reacted with a substrate to yield an intensely coloredproduct that can be analyzed with an ordinary light microscope. Whilethe list of enzyme substrates is extensive, alkaline phosphatase (AP)and horseradish peroxidase (HRP) are the two enzymes used mostextensively as labels for protein detection. An array of chromogenic,fluorogenic and chemiluminescent substrates is available for use witheither enzyme, including DAB or BCIP/NBT, which produce a brown orpurple staining, respectively, wherever the enzymes are bound.

Reaction with DAB can be enhanced using nickel, producing a deeppurple/black staining. Fluorescent reporters are small, organicmolecules used for IHC detection and traditionally include FITC, TRITC,and AMCA, while commercial derivatives, including the Alexa Fluors andDylight Fluors, show similar enhanced performance but vary in price. Forchromogenic and fluorescent detection methods, densitometric analysis ofthe signal can provide semi- and fully quantitative data, respectively,to correlate the level of reporter signal to the level of proteinexpression or localization.

The direct method is a one-step staining method and involves a labeledantibody (e.g., FITC-conjugated antiserum reacting directly with theantigen in tissue sections. While this technique utilizes only oneantibody and therefore is simple and rapid, the sensitivity is lower dueto little signal amplification, in contrast to indirect approaches.

The indirect method involves an unlabeled primary antibody (first layer)that binds to the target antigen in the tissue and a labeled secondary(second layer) that reacts with the primary antibody. As disclosed,herein, the secondary antibody must be raised against the IgG of theanimal species in which the primary antibody has been raised. Thismethod is more sensitive than direct detection strategies because ofsignal amplification due to the binding of several secondary antibodiesto each primary antibody if the secondary antibody is conjugated to thefluorescent or enzyme reporter.

Further amplification can be achieved if the secondary antibody isconjugated to several biotin molecules, which can recruit complexes ofavidin-streptavidin, or NeutrAvidin protein bound-enzyme. The differencebetween these three biotin-binding proteins is their individual bindingaffinity to endogenous tissue targets leading to nonspecific binding andhigh background; the ranking of these proteins based on theirnonspecific binding affinities, from highest to lowest, is: 1) avidin,2) streptavidin and 3) Neutravidin protein.

The indirect method, aside from its greater sensitivity, also has theadvantage that only a relatively small number of standard conjugated(labeled) secondary antibodies needs to be generated. For example, alabeled secondary antibody raised against rabbit IgG, which can bepurchased “off the shelf,” is useful with any primary antibody raised inrabbit. With the direct method, it would be necessary to label eachprimary antibody for every antigen of interest.

After immunohistochemical staining of the target antigen, a second stainis often applied to provide contrast that helps the primary stain standout. Many of these stains show specificity for discrete cellularcompartments or antigens, while others will stain the whole cell. Bothchromogenic and fluorescent dyes are available for IHC to provide a vastarray of recompounds to fit every experimental design, and include:hematoxylin, Hoechst stain, and DAPI are commonly used.

(v) Immunofluorescent Detection

As used herein, the term “immunofluorescence” refers to a technique usedfor light microscopy with a fluorescence microscope is used primarily onmicrobiological samples. This technique uses the specificity ofantibodies to their antigen to target fluorescent dyes to specificbiomolecule targets within a cell, and therefore allows visualization ofthe distribution of the target molecule through the sample.Immunofluorescence is a widely used example of immunostaining and is aspecific example of immunohistochemistry that makes use of fluorophoresto visualize the location of the antibodies. Immunofluorescent detectionis described in Am J Physiol Regul Integr Comp Physiol 301(3): R632-R640(2011).

Immunofluorescence can be used on tissue sections, cultured cell lines,or individual cells, and may be used to analyze the distribution ofproteins, glycans, and small biological and non-biological molecules.Immunofluorescence can be used in combination with other, non-antibodymethods of fluorescent staining, for example, use of DAPI to label DNA.Several microscope designs can be used for analysis ofimmunofluorescence samples; the simplest is the epifluorescencemicroscope, and the confocal microscope is also widely used. Varioussuper-resolution microscope designs that are capable of much higherresolution can also be used.

There are two classes of immunofluorescence techniques, primary (ordirect) and secondary (or indirect). Primary, or direct,immunofluorescence uses a single antibody that is chemically linked to afluorophore. The antibody recognizes the target molecule and binds toit, and the fluorophore it carries can be detected via microscopy. Thistechnique has several advantages over the secondary (or indirect)protocol below because of the direct conjugation of the antibody to thefluorophore. This reduces the number of steps in the staining proceduremaking the process faster and can reduce background signal by avoidingsome issues with antibody cross-reactivity or non-specificity. However,since the number of fluorescent molecules that can be bound to theprimary antibody is limited, direct immunofluorescence is less sensitivethan indirect immunofluorescence.

Secondary, or indirect, immunofluorescence uses two antibodies; theunlabeled first (primary) antibody specifically binds the targetmolecule, i.e. SMARCA4, and the secondary antibody, which carries thefluorophore, recognizes the primary antibody and binds to it. Multiplesecondary antibodies can bind a single primary antibody. This providessignal amplification by increasing the number of fluorophore moleculesper antigen. This protocol is more complex and time consuming than theprimary (or direct) protocol above, but it allows more flexibilitybecause a variety of different secondary antibodies and detectiontechniques can be used for a given primary antibody.

(b) Detection of Mutations Through Proteomic Analysis

Mass spectrometry-based proteomics can be employed to detect SMARCA4protein mutations and post-translational modifications (PTM). Recenttechnological advances allow thousands of proteins to be routinelyidentified from submilligram quantities of tissues or cells. Moreimportant, targeted-proteomics with prespecified protein candidates havebeen used to study protein mutation and PTM in a variety of proteins.Increased depth of proteome coverage lends itself to the investigationof molecular signatures. For instance, in cancer, thousands of mutationshave been identified but the precise relationship between genomicvariation and cancer phenotype remains largely unclear. Individualmutations may bring about proteomic changes that otherwise would not bepredicted based on known gene function.

Several choices of instrumentation exist for proteomics-based analysis.The first, shotgun proteomics, employs liquid chromatography-tandem massspectrometry (LC-MS/MS) and provides a nondirected, global inventory ofproteomes, together with quantitative assessments of protein abundances.The second protcomics approach is targeted analysis of individualproteins by multiple reaction monitoring (MRM) mass spectrometryintensity measurement of their constituent peptides.

In a typical experiment, a cancer cell and a non-cancer control cell arecultured separately. Cells are then lysed, and cellular total protein iscollected. In some instances, this protein mixture undergoes furtherpurification, e.g. immunoprecipitation to isolating certain proteins(s)or isoelectric focusing electrophoresis. However, because the improvedequipment sensitivity, this may not be required. Usually proteins arethen digested (e.g., trypsin digestion) and the resultant peptides arelyophilized to be used for mass spec analysis.

LC-MS/MS shotgun proteomic analyses can be performed on massspectrometer (e.g., an LTQ XL by Thermo Fisher Scientific) equipped witha nanospray source. For example, peptides from a protein mixturecontaining SMARCA4 can be separated on a packed capillary tip (PolymicroTechnologies, 100 mm×11 cm) with Jupiter C18 resin (5 mm, 300 Å,Phenomenex) using an in-line solid-phase extraction column (100 mm×6 cm)packed with the same C18 resin using a flit generated with liquidsilicate Kasil 1.18 Mobile phase A consisted of 0.1% formic acid andmobile phase B consisted of 0.1% formic acid in 90% acetonitrile. A90-min gradient is carried out with a 30-min washing period (100% A) toallow for solid-phase extraction and removal of any residual salts.Following the washing period, the gradient is increased to 25% B by 35min, followed by an increase to 90% B by 50 min and held for 9 minbefore returning 95% A. MS/MS spectra of the peptides are acquired usingdata-dependent scanning in which one full MS spectrum (mass range400-2000 m/z) is followed by five MS/MS spectra. MS/MS spectra arerecorded using dynamic exclusion of previously analyzed precursors for60 s with a repeat of 1 and a repeat duration of 1. MS/MS spectra weregenerated by collision-induced dissociation of the peptide ions atnormalized collision energy of 35% to generate a series of b- and y-ionsas major fragments. Biological samples from 3 independent cell cultureswere injected in duplicate for a total of 6 replicate measurements for acancer cell culture and a non-cancer cell culture.

Data from LC-MS/MS can be analyzed by a variety of methods by thoseskilled in the art. For example, MS/MS scans are transferred to mzMLfile. The resulting mzML files are then searched against the Human IPIdatabase using commercially available software. The database search isconfigured to look for both fully tryptic and semitryptic peptides witha precursor mass/charge (m/z) tolerance of 1.25 and a fragment m/ztolerance of 0.5. Carboxamidomethylation of cysteines is included asstatic modification, and oxidation of methionine as a dynamicmodification in the search criteria, while any number of missedcleavages was allowed. The IDpicker algorithm is used to assemble theset of peptides identified into a minimal list of proteins that couldexplain the observed spectral data set. A minimum of two peptides perprotein is required for valid protein identification with a peptidefalse discovery rate (FDR) of 5%. Statistically significant differencesin protein spectral counts between different groups (i.e., tumor cellsand normal cells from a SCCOHT patient) are calculated usingcommercially available software.

Similarly, targeted proteomics can be done with LC-MRM-MS. Cell linesamples for LC-MRM-MS are prepared as outlined above for LC-MS/MSproteomics, except peptide extracts are not subjected to furtherfractionation by IP or IEF. Peptide samples from each cell line areresuspended in 0.1% formic acid at 0.25 μg/μL and analyzed in triplicate(2 μL injection volume) on a triple quadrupole mass spectrometer (e.g.TSQ Vantage from Thermo-Fisher) equipped with a nanospray source. Themobile phase consists of 0.1% formic acid in either HPLC grade water (A)or 90% acetonitrile (B). An 80-min gradient is carried out with a 15-minwashing period (100% A). Following the washing period, the gradient isincreased to 60% B by 43 min, followed by an increase to 95% B by 49 minand held for 11 min before returning 97% A. Usually, a stable isotopelabeled peptide (e.g. p3-actin peptide GYSFTTTAER) is used as aninternal standard (60 fmol/injection) for relative quantification oftarget proteins by the Labeled Reference Peptide (LRP) method. Theintegrated chromatographic peak areas for the transitions of eachtargeted peptide were obtained from Skyline, and summed, normalized tosummed peak areas for the β-actin internal standard.

For either LC-MS/MS or LC-MRM-MS, peptide sequence of SMARCA4 proteinwill be obtained for both cancer cell sample and non-cancer normal cellsample. Comparison of SMARCA4 peptide sequence between the two samples,and comparing both against reference sequences, can detect somatic andgermline mutations in SMARCA4 amino acid sequence. Further analysis ofthe sequence can detect mutations for insertion, deletion or pointmutations.

3. Methodologies for Measuring SMARCA4 Gene Expression

Reduced expression of the SMARCA4 gene results in reduced cellularavailability of SMARCA4 and, as reported herein, causes growth a cancercell. Because of this and because SMARCA4 loss of expressioncharacterizes SCCOHT, expression of SMARCA4 can be used a diagnosticmarker for SCCOHT.

SMARCA4 gene expression levels (i.e., mRNA levels) can be measured byemploying such methodology as real-time PCR and electrophoresis. SMARCA4protein levels can be readily measured by ELISA, western blot, andproteomics. SMARCA4 expression can also be quantified in a tumor samplefrom a cancer patient through IHC and PCR.

In addition to the expression of SMARCA4, the same methods of measuringcellular gene and protein levels can also be to evaluate SMARCA4downstream target genes including CDH1, CDH3, EHF, RRAD and ML-IAP.

4. Methodologies for Measuring SMARCA4 Complex Formation

SMARCA4 may exert its effects on cancer cell proliferation through itsparticipation in the protein-protein complexes such as SWI/SNF and BAFcomplexes. Therefore, in addition to SMARCA4 expression, cellular levelsof downstream SWI/SNF and BAF can also be used as diagnostic factors forSCCOHT.

Protein-protein complexes can be quantified by a variety of methods bythose skilled in the art, including ELISA with antibodies recognizingdifferent components of the complex, preferably with one antibodyrecognizing SMARCA4 in the complex. Protein-protein complexes can alsobe measured in situ by molecular imaging methods usingimmunofluorescent- or immunoradioactive-probes. Double or triplestaining with such probes can be analyzed by standard imaging softwareto quantify the cellular or tissue levels of intact protein-proteincomplexes such as SWI/SNF and BAF.

Additionally, protein-protein complexes can be detected and quantifiedin cell culture or in a tumor tissue sample with fluorescence resonanceenergy transfer (FRET) which relies on the proximity of twoimmunofluorescent probes, and can be performed by those skilled in theart.

Methods for Inhibiting the Growth of an SCCOHT Cancer Cell and for theTreatment of a Patient Afflicted with Ovarian Cancer

The present disclosure provides methods for inhibiting the growth ofcancer cells and for the treatment of cancers, including ovariancancers, such as small cell carcinomas of the ovary (SCCO), inparticular SCCO hypercalcemic type (SCCOHT), which cancers areassociated with a cancer cell exhibiting reduced or undetectable SMARCA4gene expression and/or with reduced or undetectable SMARCA4 proteinlevels and/or protein function, which methods include contacting acancer cell or administering to patient afflicted with a cancer, such asan ovarian cancer, in particular SCCOHT, one or more compounds,including one or more polynucleotides, polypeptides, and/or smallmolecules, such as, for example, one or more EZH2 (histonemethyltransferase enhancer of Zeste homolog 2) inhibitors (Such asGSK-343, EPZ-6438—additional such inhibitors such as GSK 126 aredescribed in the literature or can be identified using methodology ofe.g., Garapathy—Rao et al. Chem. Biol. 2013, November 21; 20(11):1329-39incorporated herein by reference in its entirety for all purposes andQi, W. et al PNAS, 2013, www.pnas.org/cgi/doi/10.1073/pnas.1210371110incorporated herein by reference in its entirety for all purposes) orbromodomain inhibitors (Miller, S. Med. Chem Commun. 2014, 5, 288-296incorporated herein by reference in its entirety for all purposes) thatcan restore SMARCA4 gene expression and/or SMARCA4 protein levels and/orprotein function and/or can otherwise inhibit the tumorigenic effect ofa SMARCA4 gene mutation, or events triggered thereby, thereby, slowingor stopping the growth of the cancer cell. Indeed it is hypothesized andanticipated in light of evidence and experiments described herein thatreduction or elimination of SMARCA4 renders these tumors more sensitiveto such drugs. Exemplary EZH2 inhibitor compounds which are commerciallyavailable (e.g., from Active Biochem, or Selleckchem or Epizyme andother such specialty suppliers) are disclosed below:

Exemplary Structures of EZH2 Small Molecule Inhibitors Identified byGarapaty-Rao (Chem Biol. 2013 Nov. 21; 20(11): 1329-39)

As described herein, mutations, including loss-of-function mutations,within the SMARCA4 gene were identified in tissue samples from SCCOHTpatients. Those mutations were found to cause the SMARCA4 gene to eitherencode reduced or undetectable levels of SMARCA4 mRNA, to encode SMARCA4mRNA that cannot be transcribed into SMARCA4 protein, and/or to encodemRNA that does not encode functional SMARCA4 protein. Such SMARCA4 genemutations include insertion mutations, deletion mutations, frame shiftmutations, splice site mutations, nonsense mutations, and missensemutations, which result in reduced or undetectable levels of SMARCA4protein or SMARCA4 protein having reduced or undetectable functionality.For example most of the mutations characterized in the tissue samplesfrom the 12 SCCOHT patients studied included non-sense, frameshift, andslice site mutations all of which were predicted to generatenon-functional carboxy-terminal truncations of the SMARCA4 proteinlacking in one or more critical domains for protein functionalityincluding, for example the SNF2_N and helicase domains that areessential for SMARCA4 functionality.

Based in part upon these observations, the present disclosure providestherapeutic regimen that in some embodiments employ one or morecompound, such as a small molecule, polynucleotide, or polypeptide thatwhen contacted with a cell from a SCCOHT patient or when administered invivo to a SCCOHT patient is capable of restoring the expression of oneor both SMARCA4 alleles and or restoring levels and/or functionality ofSMARCA4 protein.

Thus, the present disclosure provides compounds and compositions,including pharmaceutical compositions, containing those compounds, whichcompounds and compositions may be advantageously employed in thepresently-disclosed methods for inhibiting the growth of cancer cellsand for the treatment of cancers, including ovarian cancers, such assmall cell carcinomas of the ovary (SCCO), in particular SCCOhypercalcemic type (SCCOHT), which cancers are associated with a cancercell exhibiting reduced or undetectable SMARCA4 gene expression and/orwith reduced or undetectable SMARCA4 protein levels and/or proteinfunction. Exemplified herein are compounds, including polynucleotides,polypeptides, and small molecules that can be used to restore SMARCA4gene expression and/or function and/or restore SMARCA4 protein leveland/or function and, thereby, slow or stop the growth of a cancer cell.

A wide variety of methodologies are known in the art that can be adaptedadvantageously to permit at least the partial restoration of SMARCA4gene and/or protein functionality. For example, the present disclosurecontemplates that suitable therapeutic regimen can include gene therapy,protein therapy, cell therapy in addition to or in place of traditionaldrug therapies, including conventional chemotherapies, such asintraperitoneal (IP) chemotherapy (e.g., carboplatin, paclitaxel(Taxol), liposomal doxorubicin (Caelyx, Myocet or Doxil), Gemcitabine,cisplatin, topotecan, etoposide, cyclophosphamide)), immunotherapies(e.g., bevacizumab (Avastin)), and/or radiation therapies. Thesetherapies can be used independently or together with one or more ezh2inhibitors and/or one or more JQ1 bromodomain inhibitors, in addition toand/or instead of one or more traditional cancer treatments such aschemotherapy, immunotherapy, and radiation therapy.

Three types of CRISPR/Cas systems have been described (Makarova et al.,Nat. Rev. Microbiol. 9, 467 (2011); Makarova et al., Biol. Direct 1, 7(2006); Makarova et al., Biol. Direct 6, 38 (2011)). Recent work hasshown that Type II CRISPR/Cas systems can be engineered to directtargeted double-stranded DNA breaks in vitro to specific sequences byusing a single “guide RNA” with complementarity to the DNA target siteand a Cas9 nuclease (Jinek et al., Science 2012; 337:816-821). Thistargetable Cas9-based system also works efficiently in cultured humancells (Mali et al., Science. 2013 Feb. 15; 339(6121):823-6; Cong et al.,Science. 2013 Feb. 15; 339(6121):819-23) and in vivo in zebra fish(Hwang and Fu et al., Nat Biotechnol. 2013 March; 31(3):227-9) forinducing targeted alterations into endogenous genes. Recently, it hasbeen reported to have been successful in mice (Yin, H. et al, NatureBiotechnology 32, 551-553 (2014); doi:10.1038/nbt.2884). these methodscan be used to correct the defects in SMARCA4 genes.

Further work described in U.S. Published Patent Applications20140377868; 20140357530; 20140356958; 20140342457 and 20140356959involves targeting specific sequences in eukaryotic cells in vivo inorder to alter their sequence and/or upregulating their expression.

It is contemplated that nucleic acids, in particular polynucleotidesincluding cDNAs, which encode the wild-type SMARCA4 protein can beemployed to complement the defective SMARCA4 gene. Such gene therapyapproaches include delivery technologies that permit the targeting,cellular uptake, and expression of a wild-type SMARCA4 cDNA. Suchdelivery technologies include, for example, retroviral vectors,adeno-viral vectors, a vaccinia-viral vectors, liposomes, and/ornano-particles that containing SMARCA4 gene.

The present disclosure also contemplates that wild-type SMARCA4 proteincan be delivered directly to a cell through the targeting of one or moreliposomes or a nanoparticles that contain a SMARCA4 protein alone or incombination with a targeting moiety that facilitates the delivery of theSMARCA4 protein payload to the desired cells, in particular to ovariancarcinoma cells, including SCCOHT cells.

Alternatively cells can be generated by employing recombinant DNAtechnology to engineer them to express a wild-type SMARCA4 protein.Within certain aspects, the nucleic acid encoding a wild-type SMARCA4protein can be modified by the addition of a secretory signal,regulatory sequence, and targeting moieties so that the SMARCA4 proteincan be specifically targeted to the SCCOHT cells, so that the expressionof the SMARCA4 protein can be regulated to ensure that it is deliveredat an appropriate time or when it reaches a desired target site, and sothat the SMARCA4 protein can exit the cell by exploiting nativeintracellular protein transport machinery.

Within still further aspects, the present disclosure provides smallmolecules, antibodies, antisense, shRNA, siRNA, and other RNAi-basedtechnologies that permit the delivery of target specific molecules thatcan promote the up or downregulation of genes and/or mRNAs of interest.By way of example, not limitation, it has been observed that theexpression of SMARCA2 and SMARCA4 are closely associated and that thedown-regulation of SMARCA2 can promote the up-regulation of SMARCA4.Thus, it is contemplated that suitable therapeutics for enhancing theexpression of SMARCA4 can include such small molecules, antibodies,antisense, shRNA, siRNA, and other RNAi-based technologies thatfacilitate the downregulation of SMARCA2.

In a similar fashion, it is well known that wild-type SMARCA4 plays aprimary role in regulating the expression of downstream genes including,for example, the CDH1, CDH3, EHF, RRAD, and ML-IAP genes. Thus, thepresent disclosure contemplates therapeutics, including small molecule,polynucleotide, and polypeptide therapeutics that can complement thelost gene regulatory function of mutated SMARCA4 in ovarian cancercells, especially SCCOHT cells by facilitating the upregulation of theCDH1, CDH3, EHF, RRAD, and/or ML-IAP genes. Exemplary suitable suchcompounds include small molecules, antibodies, antisense, shRNA, siRNA,and other RNAi technologies that can modulate the expression of one ormore genes that encode factors that can be exploited to induce theupregulation of the CDH1, CDH3, EHF, RRAD, and/or ML-IAP genes.

Therapeutic compounds can be administered according to therapeuticprotocols well known in the art. It will be apparent to those skilled inthe art that the administration of the disclosed therapeutic compoundcan be varied depending on the disease being treated and the knowneffects of such therapeutic compounds on that disease. Also, inaccordance with the knowledge of the skilled clinician, the therapeuticprotocols (e.g., dosage amounts and times of administration) can bevaried in view of the observed effects of the administered therapeuticcompounds on the patient, and in view of the observed responses of thedisease to the administered therapeutic compounds.

The amount of a therapeutic compound that will be effective in thetreatment, inhibition, and/or prevention of a cancer associated withdysfunctional or deficient SMARCA4 can be determined by standardclinical techniques. In vitro assays may optionally be employed to helpidentify optimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the condition being treated. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

The compounds or compositions of the present disclosure can be tested invitro, and then in vivo for the desired therapeutic or prophylacticactivity, prior to use in humans. For example, in vitro assays todemonstrate the therapeutic or prophylactic utility of aSMARCA4-promoting therapeutic compound include the effect of thetherapeutic compound on a cell line or on a patient tissue sample,wherein the compound restores a direct or indirect function of SMARCA4.The effect of a SMARCA4-promoting therapeutic compound on the cell lineand/or tissue sample can be determined utilizing techniques known tothose of skill in the art including, but not limited to proliferationand survival assays. In accordance with the present disclosure, in vitroassays that can be used to determine whether administration of aspecific SMARCA4-promoting therapeutic compound is indicated, include invitro cell culture assays in which a patient tissue sample is grown inculture, and exposed to or otherwise administered a cytotoxic compound,and the effect of such SMARCA4-promoting therapeutic compound upon thetissue sample is observed.

The present disclosure provides methods of treatment and inhibition byadministration to a patient of an effective amount of aSMARCA4-promoting therapeutic compound or composition as describedherein. In one aspect, the SMARCA4-promoting therapeutic compound issubstantially purified such that the compound is substantially free fromsubstances that limit its effect or produce undesired side-effects.

Various delivery systems are known and can be used to administer acomposition of the present disclosure, for example, encapsulation inliposomes, microparticles, microcapsules, receptor-mediated endocytosis(see, e.g., Elsabahy et al., Current Drug Delivery 8(3):235-244 (2011)for a general description of viral and non-viral nucleic acid deliverymethodologies, Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), and thelike as will be known by one of skill in the art.

Liposomal delivery methodologies are described in Metselaar et al., MiniRev. Med. Chem. 2(4):319-29 (2002); O'Hagen et al., Expert Rev. Vaccines2(2):269-83 (2003); O'Hagan, Curr. Drug Targets Infjct. Disord.1(3):273-86 (2001); Zho et al., Biosci Rep. 22(2):355-69 (2002); Chikhet al., Biosci Rep. 22(2):339-53 (2002); Bungener et al., Biosci. Rep.22(2):323-38 (2002); Park, Biosci Rep. 22(2):267-81 (2002); Ulrich,Biosci. Rep. 22(2):129-50; Lofthouse, Adv. Drug Deliv. Rev. 54(6):863-70(2002); Zhou et al., J. Inmunmunother. 25(4):289-303 (2002); Singh etal., Pharm Res. 19(6):715-28 (2002); Wong et al., Curr. Med. Chem.8(9):1123-36 (2001); and Zhou et al., Immunonmethods (3):229-35 (1994).

Nanoparticle delivery methodologies, including gold, iron oxide,titanium, hydrogel, and calcium phosphate nanoparticle deliverymethodologies, are described in Wagner and Bhaduri, Tissue Engineering18(1): 1-14 (2012) (describing inorganic nanoparticles); Ding et al.,Mol Ther e-pub (2014) (describing gold nanoparticles); Zhang et al.,Langmuir 30(3):839-45 (2014) (describing titanium dioxidenanoparticles); Xie et al., Curr Pharm Biotechnol 14(10):918-25 (2014)(describing biodegradable calcium phosphate nanoparticles); Sizovs etal., J Am Chem Soc 136(1):234-40 (2014) (describing sub-30 monodisperseoligonucleotide nanoparticles).

Herpes Simplex Virus vectors for the delivery of nucleic acids to targetcells have been reviewed in Anesti and Coffin, Expert Opin Biol Ther10(1):89-103 (2010); Marconi et al., Adv Exp Med Biol 655:118-44 (2009);and Kasai and Saeki, Curr Gene Ther 6(3):303-14 (2006). Lentiviralvectors for the delivery of nucleic acids to target cells have beenreviewed in Primo et al., Exp Dermatol 21(3):162-70 (2012); Staunstrupand Mikkelsen, Curr Gene Ther 11(5):350-62 (2011); and Dreyer, MolBiotechnol 47(2): 169-87 (2011). Adenovirus vectors for the delivery ofnucleic acids to target cells have been reviewed in Huang and Kamihira,Biotechnol Adv. 31(2):208-23 (2013); Alemany, Adv Cancer Res 115:93-114(2012); Kaufmann and Nettelbeck, Trends Mol Med 18(7):365-76 (2012); andMowa et al., Expert Opin Drug Deliv 7(12):1373-85 (2010).Adeno-associated virus (AAV) vectors for the delivery of nucleic acidsto target cells have been reviewed in Li et al., J. Control Release172(2):589-600 (2013); Hajitou, Adv Genet 69:65-82 (2010); McCarty, MolTher 16(10):1648-56 (2008); and Grimm et al., Methods Enzymol392:381-405 (2005).

Methods of systemic administration include, but are not limited to,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, and oral routes. The SMARCA4-promoting therapeutic compoundsor compositions may be administered by any convenient route, for exampleby infusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other therapeuticallyeffective compounds. Pulmonary administration can also be employed, forexample, by use of an inhaler or nebulizer, and formulation with anaerosolizing compound.

Local modes of administration are also contemplated. For example, it maybe desirable to introduce the SMARCA4-promoting therapeutic compounds orcompositions into the tumor by any suitable means, including in situdepot administration or injection. In situ depot administration may befacilitated by an intraventricular catheter, for example, attached to areservoir, such as an Ommaya reservoir. Other localized administrationmethods include without limitation local infusion during surgery,topical application, by injection, by means of a an implant, saidimplant being for example made from a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.

The SMARCA4-promoting therapeutic compounds can be delivered in avesicle, such as a liposome (Langer, Science 249:1527-1533 (1990)) or ina controlled release system. A controlled release system can be placedin proximity of the therapeutic target, thus requiring only a fractionof the systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, Vol. 2, pp. 115-138 (1984)).

It will be understood that, unless indicated to the contrary, termsintended to be “open” (e.g., the term “including” should be interpretedas “including but not limited to,” the term “having” should beinterpreted as “having at least,” the term “includes” should beinterpreted as “includes but is not limited to,” etc.). Phrases such as“at least one,” and “one or more,” and terms such as “a” or “an” includeboth the singular and the plural.

It will be further understood that where features or aspects of thedisclosure are described in terms of Markush groups, the disclosure isalso intended to be described in terms of any individual member orsubgroup of members of the Markush group. Similarly, all rangesdisclosed herein also encompass all possible sub-ranges and combinationsof sub-ranges and that language such as “between,” “up to,” “at least,”“greater than,” “less than,” and the like include the number recited inthe range and includes each individual member.

All references cited herein, whether supra or infra, including, but notlimited to, patents, patent applications, and patent publications,whether U.S., PCT, or non-U.S. foreign, and all technical and/orscientific publications are hereby incorporated by reference in theirentirety.

While various embodiments have been disclosed herein, other embodimentswill be apparent to those skilled in the art. The various embodimentsdisclosed herein are for purposes of illustration and are not intendedto be limiting, with the true scope and spirit being indicated by theclaims.

The present disclosure will be further described with reference to thefollowing non-limiting examples. The teaching of all patents, patentapplications and all other publications cited herein are incorporated byreference in their entirety.

EXAMPLES Example 1 Unbiased Screening of 279 Cancer Genes IdentitiesSMARCA4 as a Novel Gene Underlying Small Cell Ovarian Cancer

This Example demonstrates that an unbiased screening of 279 cancer genesin 12 ovarian cancer patients identifies the gene coding SMARCA4protein, a member of several key tumor suppressor complexes, as a novelmarker for a variant of ovarian cancer, namely small cell carcinoma ofthe ovary, hypercalcemic type (SCCOHT). Mutations of SMARCA4 weredetected in 100% of patients examined, making SMARCA4 an excellentdiagnostic marker for this type of rare and aggressive ovarian canceraffecting young women.

SCCOHT is a rare, aggressive form of ovarian cancer diagnosed in youngwomen that is generally fatal when spread beyond the ovary. Mostpatients relapse and die within 2 years of diagnosis, regardless ofstage, with a long-term survival rate of only 33%, even when disease isconfined to the ovary at diagnosis. Seidman et al., Gynecol Oncol 59:283-7 (1995). Thus, a genetic marker is urgently needed for the earlydiagnosis and treatment of this aggressive disease.

Tissue samples from twelve patients that had been diagnosed with SCCOHTwere reviewed by a specialty gynecologic pathologist to confirm thediagnosis of SCCOHT, which is characterized by a highly cellular andhighly proliferative small cell malignancy with minimal stroma withfollicle-like spaces. FIG. 10 shows the typical histopathologicalfeatures of SCCOHT, including a combination of small neoplastic cellsforming a pseudofollicular space and larger rhabdoid cells, are visiblein a sample obtained from 1 of 12 tumors that were subjected to targetcapture and massively parallel DNA sequencing (hematoxylin and eosin).The presence of a large cell or rhabdoid component was accepted as partof the spectrum of this disease. The following entities were excludedfrom consideration with a combination of morphologic examination andimmunohistochemistry (IHC): small cell neuroendocrine carcinoma,juvenile granulosa cell tumor, poorly differentiated Sertoli-Leydig celltumor, desmoplastic small round cell tumor, metastatic melanoma,lymphoma, and rhabdomyosarcoma. Clinical data collection was limited toonly age and year of diagnosis due to HIPAA regulations. No familyhistory was available. Diagnosis of SCCOHT was also facilitated by IHCof common markers including EMA, p53, cytokeratin, LCA, inhibin, CD10,S100, desmin and WT1 (Table 2).

TABLE 2 Summary of Immunohistochemistry Studies Performed to Assist withDiagnostic Interpretation Case EMA P53 Cytokeratin Inhibin LCA CD10 S100Desmin WT1 101 ND ND + ND ND ND ND − + 102 − + + − − + − − ND 103 + + +− − + − − ND 104 ND ND + ND ND ND ND ND ND 105 + + ND − − − − +106 + + + − − + − ND ND 107 + ND ND − ND ND − − + 108 + + + − − + − − +109 − + − − − ND − − + 110 ND ND ND ND ND ND ND ND ND 111 + + + − ND NDND − ND 112 − ND − − − ND − − +

DNA and RNA were extracted from paired formalin-fixed, paraffin-embedded(FFPE) tumors from these 12 patients with at least 50% tumor cell nucleiand normal tissue samples according to standard protocols. Germline DNAwas derived from either peripheral lymphocytes or FFPE blocks ofanatomically distant tissues, such as benign lymph nodes, and used asthe source of normal tissue.

To identify key genes underpinning SCCOHT, paired normal and tumorsamples were sequenced to a depth of at least 100× using target captureand massively parallel sequencing. The inventors profiled genomicalterations in 279 key cancer-associated genes using IMPACT (IntegratedMutation Profiling of Actionable Cancer Targets), a custom hybridcapture-based deep sequencing assay. Won et al., J Vis Exp October18(80):e50710 (2013). The selected genes encompass all well-establishedoncogenes and tumor suppressor genes including all druggable targets ofFDA-approved therapies and investigational compounds in clinical trialsat MSKCC. Custom oligonucleotide probes were designed to capture allprotein-coding exons and select introns of these 279 commonly implicatedoncogenes, tumor suppressor genes, and members of pathways deemedactionable by targeted therapies.

Barcoded sequence libraries were prepared (New England Biolabs, KapaBiosystems) and exon capture was performed on barcoded pools byhybridization (Nimblegen SeqCap) using an input of 97-250 ng DNA, aspreviously described. Wagle et al., Cancer Discov 2:82-93 (2012).Captured pools were sequenced on an Illumina HiSeq 2000 (2×75 bp reads),and reads were aligned to the reference human genome (hg19) using theBurrows-Wheeler Alignment tool. Li and Durbin, Bioinformatics 25:1754-60(2009). Duplicate filtering, local multiple sequence alignment and basequality score recalibration were performed using the Genome AnalysisToolkit (GATK) according to GATK best practices. DePristo et al., NatGenet 41:491-8 (2011). Sequence data were analyzed to identify 3 classesof somatic alterations: single-nucleotide variants using MuTect,(Cibulskis et al., Nat Biotechnol 31:213-9 (2013)) small indels usingSomaticIndelDetector (DePristo et al., Nat Genet 43:491-8 (2011)), andcopy number alterations, as previously described. Wagle et al., CancerDiscov 2:82-93 (2012). All candidate mutations and indels were manuallyreviewed using the Integrative Genomics Viewer. Robinson et al., NatBiotechnol 29:24-6 (2011). All mutations were validated using Sangersequencing in both genomic DNA and RNA transcripts (cDNA) to confirm thesomatic nature of the alteration and transcript expression. Primersspanning exon/intron 18, 24 and 28 were constructed for cases 101, 102and 112, respectively, to determine the presence of retained introns.For cDNA synthesis, the SuperScript III One-Step RT-PCR System(Invitrogen) was used according to the manufacturer's instructions.

Computational and biostatical analyses were performed. Mutationfrequencies across The Cancer Genome Atlas (TCGA) tumor types werecollated from data contained within the cBioPortal for Cancer Genomics(http://cbioportal.org). Cerami et al., Cancer Discov 2:401-4 (2012);Gao et al., Sci Signal 6:11 (2013). Background mutation frequencies forthe 279 genes sequenced as part of this study were also obtained forTCGA tumor types, excluding hypermutated cases that carry more than1,000 nonsynonymous mutations.

Results from target capture and massively parallel sequencing revealedthat SMARCA4 mutations occurred throughout various exons and includednonsense, frameshift, and splice site mutations as well as a homozygousintragenic deletion of two exons summarized in FIG. 1A. No SMARCA4missense mutations were identified. Sequence variants from all 12 caseswere further validated using Sanger sequencing. cDNA was sequenced inseven samples, and all were found to have mutations expressed within RNAtranscripts. Next-generation sequencing data and sequence-specificelectropherograms were obtained for all 12 cases (data not shown).

One case contained a germline mutation (case 111, Table 1), which isconsistent with prior reports suggesting a hereditary component to thisdisease. Longy et al., J Med Genet 33:333-5 (1996); McDonald et al. JPediatr Surg 47:588-92 (2012).

The probability of finding one gene mutated in all 12 samples when 279genes are sequenced is given by 1-(1-p12)279. This assumes that mutationof a given gene in a patient is a Bernoulli trial with probability p,the gene mutations are exchangeable, and also independently andidentically distributed across patients. We used p=0.015, derived fromTCGA samples as explained above. The tumor and normal samples weresequenced to a mean depth of 442× across all genes. A minimum depth of100× was achieved in 97% of target exons in tumors. The SMARCA4 somaticmutations identified in all tumor samples are shown in Table 1.

In conclusion, among all 279 cancer-related genes screened among the 12SCCOHT cases, one gene remarkably stood out: SMARCA4 mutations wereidentified in each case (FIG. 1B). The probability of identifyingSMARCA4 mutations in all 12 samples is less than 2.22×10⁻¹⁶. Only fouradditional non-recurrent somatic mutations were identified in any of theother 278 genes sequenced across all 12 samples. In contrast, ananalysis of 4,784 non-hypermutated tumors across The Cancer Genome Atlas(TCGA) revealed somatic mutations in an average of 4.3 of these 279genes (STD 4.4) per tumor. TCGA samples with inactivating SMARCA4mutations had more mutations in the other 278 genes sequenced (mean=14)in contrast to the SCCOHT cases. Thus, SMARCA4 is a novel marker forovarian cancer, especially SCCOHT.

Example 2 Mutations that Reduce SMARCA4 Gene Expression and/or SMARCA4Protein Expression are Associated with SCCOHT

This Example demonstrates that the SMARCA4 mutations identified among 12SCCOHT patients in Example 1 are all loss-of-function mutations, thussuggesting SMARCA4 may be causatively linked to ovarian cancer,especially SCCOHT.

Examination of the SMARCA4 mutations demonstrated that they occurredthroughout various exons and included nonsense, frameshift, and splicesite mutations as well as a homozygous intragenic deletion of two exons.FIG. 1A summarizes the mutations among all 12 SCCOHT patients.Interestingly, no SMARCA4 missense mutations were identified in these 12patients.

To confirm the results obtained from IMPACT, sequence variants from all12 cases were validated using Sanger sequencing. cDNA was sequenced in 7samples, and all were found to have mutations expressed within RNAtranscripts. Next-generation sequencing data and sequence-specificSanger electropherogram validation results were obtained for all 12cases. 4 cases harbored two inactivating mutations each in SMARCA4(cases 104, 105, 106, 109). The remaining eight cases harbored singleinactivating mutations accompanied by loss of heterozygosity at theSMARCA4 locus (supported by adjacent single nucleotide polymorphisms[SNPs]); in each case, the mutant allele frequency was 0.75 or greater.

Due to the precise location of biallelic splice site mutations withinintronic sequence at the highly conserved AG donor region, we testedwhether introns in cases 101, 102, and 112 were retained. We identifiedpreferential intronic expression, as expected, in cDNA sequenced from arepresentative tumor sample with a splice site mutation (case 102, FIG.6A). Onestep RT-PCR confirmed continuation of transcription downstreamfrom the splice site mutations, suggesting that the splice sitemutations do not cause mRNA truncation (case 102, FIG. 6B).

A homozygous in-frame deletion of 102 amino acids from exons 25 and 26detected in case 103 was confirmed by Sanger sequencing of cDNA, whichresulted in partial deletion of the helicase domain. One-step RT-PCRconfirms that tumor tissue yields a single band with primers that spanexons 24 and 27 (FIG. 5A). Upstream and downstream primer pairsconfirmed that transcription continued downstream of this deletion (FIG.5B). IHC showed the retention of protein expression. However, sequencingdata confirming an impaired C-terminal helicase domain suggests thatthis deletion results in translation of a truncated non-functionalcatalytically dead product. A homozygous in-frame four amino acid (ETVN)deletion within exon 27 was detected in case 107. No additional tissuewas available to demonstrate the effect on protein expression.

In addition to the 12 SCCOHT patients, the inventors also examined theassociation of SMARCA4 mutations and its expression in other types ofcancers. FIG. 3 shows grouped data demonstrating SMARCA4 gene expressionacross TCGA tumors for cases with available mutation and RNA sequencedata (RSEM). A correlation is seen between inactivating SMARCA4mutations and decreased gene expression across various solid tumors.

In summary, among all 12 patients (Table 1), inactivating biallelicmutations of SMARCA4 gene are found in each case. Inactivating mutationsinclude insertions and deletions, frame shift, splice site and nonsensemutations. The SCCOHT tumors had few other mutations in the panel of 278sequenced genes. Though the study is limited by a modest samples sizedue to the rarity of this disease, the identification of inactivatingmutations in a single gene in all 12 tumors studied is consistent withthe characteristics of a tumor suppressor. Most of the identifiedmutations reside within the known helicase catalytic domains of SMARCA4,suggesting a potential role in tumorigenesis.

Example 3 SCCOHT Patients Exhibit Dysfunctional or Deficient SMARCA4Protein

This Example demonstrates that the inactivating mutations of SMARCA4identified among 12 SCCOHT patients in Example 2 also result in the lossof protein expression and function. This observation strongly suggestsSMARCA4 is not merely a marker of the disease, but it may be causativelylinked to SCCOHT.

SMARCA4 (BRG1) immunohistochemistry (IHC) was performed in the ninecases with available tissue samples. The IHC staining method for SMARCA4was optimized using several antibodies in a variety of differentconditions until one was chosen on the basis of its ability todemonstrate consistent nuclear staining patterns in a small group ofhigh-grade serous ovarian cancers serving as positive controls. Onewhole FFPE section from each of the available SCCOHT cases was evaluatedwith a commercially available polyclonal antibody against SMARCA4(Upstate cell signaling solutions, Cat #07-478). Whole sectionsunderwent epitope retrieval using heat by steaming with EDTA at pH 8 for30 minutes. This was followed by overnight incubation with the primaryantibody at 4° C. (dilution 1:4000). Detection of bound antibody wasaccomplished with biotinylated anti-rabbit IgG (dilution 1:500, VectorLaboratories, Cat # BA-1000) and ABC (Vector Laboratories, Cat #PK-6100). DAB was used as the chromogen. For tumors with mutant SMARCA4and loss of SMARCA4 protein expression, positive staining of bloodvessels and stromal cells was used as an internal positive control. Theabsence of nuclear staining in tumor cells in the presence of aninternal positive control was scored as “loss of expression.”

High-grade serous ovarian cancer with wild-type SMARCA4 sequence servedas a positive control. In cases 104, 105, 106, 108, 109 and 110,nonsense mutations resulted in a premature stop codon within the openreading frame of the mRNA transcript. In cases 104, 105, 106 and 109,the nonsense mutation was heterozygous with a concurrent frameshift orsplice site mutation. IHC demonstrated loss of SMARCA4 nuclear stainingand retention of staining in the internal positive control cells in allfour cases with nonsense mutations that had available tissue (FIG. 4).Compared to the strong SMARCA4 staining in the positive control sample(top left panel, FIG. 4), all the cases with tumor tissue samplesprocessed by IHC showed significantly less staining intensity. Case 111was found to have a heterozygous germline nonsense mutation with loss ofthe wild-type somatic allele and associated loss of IHC proteinexpression. This case had a paucity of pseudofollicular spaces that weremore common in the remainder of the study cohort.

Immunoblotting was also used to validate the results from IHC inrepresentative patients. Frozen tumor samples were available from 2cases (101 and 102). Prior to protein extraction, cells were washed withice-cold PBS. Cells were lysed in RIPA buffer. Extracted proteins wereresolved by SDS-PAGE electrophoresis, transferred on nitrocellulose, andblotted with polyclonal anti-Brg-1 (dilution 1:1000; Santa CruzBiotechnology, Cat# sc-10768) and anti-α-tubulin (Santa CruzBiotechnology, Cat # sc-5546) as a loading control.

Both immunoblots and IHC showed clear loss of SMARCA4 protein in cases101 and 102 (FIG. 2B and FIG. 4). Case 112 had equivocal loss of IHCprotein expression with tumor cells staining less intensely than normaltissue elements. A homozygous in-frame deletion of 102 amino acids fromexons 25 and 26 detected in case 103 was confirmed by Sanger sequencingof cDNA, which resulted in partial deletion of the helicase domain.Upstream and downstream primer pairs confirmed that transcriptioncontinued downstream of this deletion. IHC showed the retention ofprotein expression. However, sequencing data confirming an impairedC-terminal helicase domain suggests that this deletion results intranslation of a truncated non-functional catalytically dead product. Ahomozygous in-frame four amino acid (ETVN) deletion within exon 27 wasdetected in case 107. No additional tissue was available to demonstratethe effect on protein expression.

In conclusion, protein studies of tumor tissue samples from SCCOHTpatients demonstrate the loss of protein expression and immunoreactivityof SMARCA4. These results are consistent with the nucleotide analysis ofSMARCA4 gene shown in Example 2. Thus, SCCOHT patients exhibit mutationsof SMARCA4 genes that result in dysfunctional or deficient SMARCA4protein, suggesting important oncogenic and therefore targetablefunctions.

Example 4 SMARCA4 Regulates Cell Proliferation. Demonstrating SMARCA4 asa Therapeutic Target

This Example demonstrates that SMARCA4 is not only a marker for SCCOHT,but its absence or dysfunction is likely to contribute to thedevelopment of SCCOHT. Functional gain-of-function and loss-of-functionstudies demonstrate that SMARCA4 is essential for regulating cellproliferation.

To determine the functional effects of SMARCA4 loss, SMARCA4 wasectopically re-introduced through electroporation in SMARCA4-null H1299human non-small cell lung adenocarcinoma cells (available as CRL-5803™from ATCC®, Manassas, Va.). SMARCA4 is not a marker for this cancer butthis cell line is SMARCA4 null. The H1299 cell line was authenticated inJune 2013 by STR DNA profiling method (Genetica DNA Laboratories) usingDSMZ database. H1299 cells were cultured in RPMI media supplemented with10% FCS. 293T cells were obtained from ATCC in September 2011. The cellswere cultured in DME-HG media supplemented with 10% FCS. All cells weretested negative for the mycoplasma. Plasmid containing SMARCA4 cDNA(pCMV6-XL5; Origene Cat# SC323288) was transfected in H1299 cells byelectroporation using Nucleofector (Amaxa). 24 h post-electroporationcells were counted using TC10 Automated Cell Counter (BioRad), proteinswere extracted and analyzed by immunoblotting.

H1299 cells lack SMARCA4 expression as validated by western blot (FIG.2B). After the introduction of SMARCA4 cDNA, SMARCA4 protein wasdetected in H1299 in a dose-dependent manner (FIG. 7A). Re-expression ofSMARCA4 resulted in a dose-dependent suppression of cell growth (FIG.7B). The expression of p21 also increased consistent with prior reportsof SMARCA4's effect on cell cycle arrest (FIG. 7A). Hendricks et al.,Mol Cell Biol 24:362-76 (2004); Napolitano et al., J Cell Sci120:2904-11 (2007).

In addition to gain-of-functions in SMARCA4-deficient cells, we alsoperformed loss-of-function studies in cells normally expressing SMARCA4.

To knock-down SMARCA4 GIPZ SMARCA4 shRNA Viral Particle Starter Kit wasused (Thermo Scientific). Transduction was performed according tomanufacturer's suggestions. A stable knock-down was obtained byselection with 2 μg/mL puromycin. XTT proliferation assay (ATCC;Cat#30-1011K) was performed according to manufacturer's suggestions.40,000 cells were seeded in 24-well dish and absorbance was measured atdifferent time points after the seeding (24-96 h) using Synergy™ HTPlate Reader (BioTek) and used to report relative cell proliferation.

The shRNA against SMARCA4 was able to significant knock down itsexpression, as validated by western blot (FIG. 7C), which led to anincrease in cell proliferation as measured by an XTT proliferation assay(FIG. 7D).

Results from these functional experiments with cultured cellsdemonstrating the crucial role of SMARCA4 in cell proliferation areconsistent with the putative functions of SMARCA4. SMARCA4 and othermembers of the SMARCA4, especially SMARCA2, are key components ofprotein-protein complexes with tumor suppressing abilities. The highlyhomologous SMARCA4 (BRG1) and SMARCA2 (BRM) proteins are mutuallyexclusive ATP-dependent catalytic subunits within the BAF complex andco-exist with ARID1A (BAF250A) or ARID1B (BAF250B). Reisman et al.,Oncogene 28:1653-68 (2009); Wilson and Roberts, Nat Rev Cancer 11:481-92(2011). SMARCA4 in the PBAF complex is associated with the PBRM1(BAF180) subunit, which is absent from the BAF complex. Recent studieshave demonstrated that the silencing of SMARCA2 through RNA interferencesuppresses growth of SMARCA4-deficient lung cancer cell lines andxenografts, suggesting a synthetic lethal relationship. Oike et al.,Cancer Res 23:5508-18 (2013). ARID1A loss has recently been shown to betumorigenic in an ovarian cancer lineage, suggesting that SMARCA4 lossmay be sufficient for transformation through BAF complex p53-dependentmechanisms. Guan et al., Cancer Res 71:6718-27 (2011). SMARCA2-ATPaseinhibitors may be an effective approach for treating SMARCA4-deficienttumors. Improved understanding of the SWI/SNF complex function andcharacteristics are now opening therapeutic opportunities for affectedtumors. Since SMARCA2 and SMARCA4 are mutually exclusive subunits, weexamined the co-occurrence of SMARCA2 and SMARCA4 inactivating mutationsamong all non-hypermutated TCGA tumors. We found only one case with aninactivating SMARCA4 mutation and a SMARCA2 missense mutation.

In conclusion, both gain-of-function and loss-of-function experimentsdemonstrate that SMARCA4 plays a critical role in regulating cellproliferation. While a decrease in SMARCA4 expression promotes cellproliferation, an increase in its expression suppresses cellproliferation. These results are consistent with the clinical data thatloss-of-function SMARCA4 mutations identified from 12 SCCOHT patientsare associated with tumor growth. Taken together, the associationbetween SMARCA4 and SCCOHT is likely a causal one; that is, loss offunction in SMARCA4 by inactivating mutations may be one cause oftumorigenesis in SCCOHT patients, and likely also plays a role in tumorgrowth and disease progression. Thus, SMARCA4 emerges as a novel targetfor intervention for the prevention and treatment of ovarian cancer,especially SCCOHT. In addition, the knowledge gained fromgain-of-function studies with SMARCA4 expression vector can also be usedto for developing gene and cell therapies that promote SMARCA4expression.

Example 5 Use of SMARCA4 to Regulate Tumor Growth

This Example describes experiments that have indicated or willdemonstrate SMARCA4 may also be used as a therapeutic or therapeutictarget for other types of cancer.

SMARCA4 mutations have been previously reported at low frequency inother solid tumors. Across all tumors characterized by TCGA to date,SMARCA4 mutations have been detected in 3% of 4,787 non-hypermutatedsamples (FIG. 1B and FIG. 10). Mutation frequencies of 5-8% are presentin bladder carcinoma, stomach adenocarcinoma, lung adenocarcinoma, andlower grade glioma. Of the 128 somatic SMARCA4 mutations in the TCGAsamples, 84% were missense variants of uncertain functional significance(FIG. 1B).

For all TCGA samples, the mean RSEM (2050, std: 1760) was less insamples with non-missense mutations than other samples without mutationsor with only missense mutations (3724, std: 1692; P=8.7×10⁻⁴) (FIG. 3).For TCGA lung adenocarcinoma samples, the mean RSEM (601, std: 370) wasless in samples with non-missense mutations than other samples withoutmutations or with only missense mutations (3330, std: 1524; P=2×10⁻⁸)(FIG. 3).

Examination of SMARCA4 in various cancers identifies that in addition toovarian cancer, especially SCCOHT, inactivating SMARCA4 mutation is alsofrequently found in lung adenocarcinoma, and lower grade glioma amongother cancers. The association between inactivating mutations in SMARCA4and lung adenocarcinoma was analyzed and it was discovered that patientswith inactivating SMARCA4 mutations exhibited poor outcome (FIG. 8).Their survival rate was significantly lower compared to patients withoutinactivating SMARCA4 mutations (p=0.02). This suggests that the role ofSMARCA4 in controlling proliferation has therapeutic implications beyondSCCOHT (where SMARCA4 is a diagnostic marker) to other cancerpopulations (even cancers where loss of SMARCA4 has an incidence too lowto serve diagnostically). A therapeutic intervention restorative ofSMARCA4 expression and function for the SMARCA4 deficient patientsubpopulation may be useful to control tumor growth and therefore reducetumor burden. Indeed, overexpression of SMARCA4 both in SMARCA4deficient and SMARCA4 competent tumors may control tumor growth.

In conclusion, the evidence linking SMARCA4 to ovarian cancer,especially SCCOHT, is strong. But the epidemiological evidence presentedhere, coupled with the observation that SMARCA4 regulates cellproliferation, suggests that SMARCA4 may be a candidate for atherapeutic target for other types of cancers, such as lungadenocarcinoma, and lower grade glioma.

Example 6 Recurrent SMARCA4 Mutations in Small Cell Carcinoma of theOvary

It has been suggested that SMARCA4 acts as a tumor suppressor. Toconfirm SMARCA4's tumor suppressor role in small cell carcinoma of theovary hypercalcemic type (SCCOHT), SMARCA4 gene has been knocked downand is planned to be knocked down in target cell lines and measuretumorigenic effects. Although it remains speculative, it has beensuggested that SCCOHT originates from ovarian epithelial cells.Therefore, as model cell lines we will use a panel of normal ovarianepithelial cell lines such as T80, HOSE 6-7, OSE. To knock-down SMARCA4,we will use the same approach as in our preliminary studies where wesuccessfully depleted SMARCA4 in T80 cells and demonstrated an increasein cell growth reflective of SMARCA4's tumor suppressor activity (FIGS.11A and 11B).

GIPZ SMARCA4 Viral Starter Kit (Thermo Scientific) provides purifiedshRNA viral particles with appropriate negative and positive controls.Target cell lines can be transduced with these particles according tomanufacturer's suggestions. Briefly, at day 0, cells can be seeded toapproximate 80% confluence and the following day the viruses can beapplied. To achieve stable knock-down and select for successfullytransduced cells, the cells can be treated with puromycin. Knock-downcan be tested on both RNA and protein levels utilizing Real-Time PCRTaqman (Applied Biosystems) and Western blotting assays, respectively.

Once knock-down is confirmed, assays that are traditionally used tomeasure malignant transformation, such as cell proliferation (XTT assayfrom ATCC), cell migration and invasion assays (BD Biosciences) can beperformed. The Soft Agar Assay can be used for Colony Formation (CellBiolabs), which is considered the most stringent assay for measuringmalignant transformation of cells. All assays can be performed accordingto manufactures' suggestions.

As shown in preliminary studies in T80 cells, cell growth increases uponSMARCA4 depletion. Given that it has been demonstrated that BIN-67cells, a SCCOHT cell line, is capable of forming spheroids (Gamwell L Fet al. Orphanet Journal of Rare Diseases; 2013 Feb. 21; 8:33) colonyformation in a Soft Agar Assay can be used to demonstrate that SMARCA4knock-down results in increased cell growth.

The BIN-67 cell line can also be used to complement knock-down studies,by showing the reduction in cell growth upon SMARCA4 re-introductioninto those cells. The lack of SMARCA4 expression can first be confirmedin those cells and SMARCA4 can be overexpressed with a plasmidcontaining SMARCA4 cDNA (pCMV6-XL5; OriGene). Over-expression can beconfirmed by western blot. In this way, it can be demonstrated thatSMARCA4 over-expression reduces cell growth and, thereby, reverses themalignant phenotype.

Example 7 A Xenograft Animal Model for Testing Potentially TherapeuticDrugs

Ongoing and future studies involve using mouse xenografts. This willpermit confirmation of SMARCA4's tumor suppressor activity in vivo aswell as to explore potential therapies. A preliminary xenografting studyhas been conducted using 293T cells depleted of SMARCA4 (FIG. 12A).Briefly, 5-6 weeks old athymic females were and will be used with n=5for each experimental group. Five million cells were and will besubcutaneously injected in both the left and right flank. Tumor growthwas and will be monitored over the course of a couple of weeks.Preliminary data (FIGS. 12A and 12B.) confirm that depletion of SMARCA4in 293T cells leads to more aggressive growth in mice, furthersupporting previously adduced data that SMARCA4 is a tumor suppressor.These studies will be expanded using more cell lines that we plan todeplete of SMARCA4 as described above.

Similarly, xenografting will be used to explore potential therapies.Several drugs could be promising for targeting SMARCA4 mutant tumors,such as EZH2 inhibitors and drugs that target DNA damage repairpathways. EZH2 hyperactivity has been explored as a therapeutic targetand several EZH2 inhibitors have been tested in preclinical and clinicalstudies. Given that SMARCA4 and EZH2 have an antagonistic relationship,and it has been suggested that SMARCA4 depletion can cause EZH2hyperactivity, we anticipate that SMARCA4-depleted tumors will besensitive to EZH2 inhibitors. In our initial studies, we will use theEZH2 inhibitors GSK126, EPZ-6438 and GSK343. We will also explore thesensitivity of SMARCA4 depleted cells to DNA damage repair targetingdrugs. The most recent studies suggest SMARCA4's role in DNA damagerepair pathways. Depletion of SMARCA4 would render cells sensitive tothe DNA damage repair pathway targeting drugs, such as PARP inhibitors(e.g. BSI 201 or BMN 673 by way of nonlimiting example).

Exemplary PARP inhibitor compounds are disclosed below:

Preliminary data in SMARCA4-null SCCOHT cell line Bin67 confirm thesensitivity to the PARP inhibitor BMN-673 and camptothecin (CPT), a DNAdamaging agent. This will be further tested in vivo using mousexenografts. We anticipate that SMARCA4-depleted cells will be sensitiveto drugs targeting DNA damage repair pathways as well as combinations ofsuch drugs with more traditional chemotherapeutic drugs. In addition,combination therapies using EZH2 inhibitors and DNA repair drugs orconventional chemotherapeutics can thus be tested.

All mouse xenografting will be performed as described above. Initially,we will use 293T cells that are either WT or SMARCA4-depleted. Drugadministration will be performed according to manufacturers' directions.For those drugs that are not already tested in mice, we will performdose-escalation studies to test for drug toxicity. The drugs thatspecifically kill SMARCA4-depleted cells will be used in future studies.

TABLE 3 Sequences Listed Sequence ID Number GenBank ID Match SEQ ID NO:1 Homo sapiens SMARCA4 isoform 1 mRNA (GenBank EU430759.1) SEQ ID NO: 2Homo sapiens SMARCA4 isoform 1 Protein (GenBank EU430759.1) SEQ ID NO: 3Homo sapiens SMARCA4 isoform 2 mRNA (GenBank EU430757.1) SEQ ID NO: 4Homo sapiens SMARCA4 isoform 2 Protein (GenBank EU430757.1) SEQ ID NO: 5Homo sapiens SMARCA4 isoform 3 mRNA (EU430756.1) SEQ ID NO: 6 Homosapiens SMARCA4 isoform 3 Protein (EU430756.1) SEQ ID NO: 7 Homo sapiensSMARCA4 isoform 4 mRNA (EU430758.1) SEQ ID NO: 8 Homo sapiens SMARCA4isoform 4 Protein (EU430758.1)

1. A method for the diagnosis of small cell carcinoma of the ovaryhypercalcemic type (SCCOHT), said method comprising detecting in abiological sample from a subject one or more mutations in a SMARCA4 geneand/or a regulatory sequence associated with the SMARCA4 gene, each ofwhich mutations is known, predicted, or demonstrated to reduce oreliminate at least one of SMARCA4 gene expression, SMARCA4 proteinlevel, and SMARCA4 protein function.
 2. The method of claim 1 wherein atleast one of said mutations in said SMARCA4 gene is a germline mutationor a somatic mutation.
 3. (canceled)
 4. The method of claim 1 whereineach allele of the SMARCA4 gene and/or associated regulatory sequencecomprises one or more mutations, each of which mutations is known,predicted, or demonstrated to reduce or eliminate at least one ofSMARCA4 gene expression, SMARCA4 protein level, and SMARCA4 proteinfunction.
 5. The method of claim 1 where each of said mutations in saidSMARCA4 gene is selected from the group consisting of an insertionmutation, a deletion mutation, a frame shift mutation, a splice sitemutation, a nonsense mutation, and a missense mutation.
 6. The method ofclaim 1 wherein at least one of said mutations in said SMARCA4 gene isupstream of the region of said SMARCA4 gene that encodes a HAS domain.7. The method of claim 1 wherein at least one of said mutations in saidSMARCA4 gene is (i) upstream of a region of said SMARCA4 gene thatencodes a BRK domain; or (ii) within a region of said SMARCA4 gene thatencodes a SNF2_N domain; or (iii) is upstream of or within a region ofsaid SMARCA4 gene that encodes a Helicase Domain; or (iv) is upstream ofor within a region of said SMARCA4 gene that encodes a SnAC Domain; or(v) is upstream of or within the region of said SMARCA4 gene thatencodes a Bromo Domain or a combination of two or more of the foregoing.8. (canceled)
 9. (canceled)
 10. (canceled)
 11. (canceled)
 12. A methodfor inhibiting the growth of a cancer cell exhibiting reduced orundetectable: (i) SMARCA4 protein level; (ii) SMARCA4 protein function,or (iii) both SMARCA4 protein levels and function, said methodcomprising contacting said cancer cell with one or more compounds thatcan restore one or more of SMARCA4 gene expression, SMARCA4 proteinlevel, and/or SMARCA4 protein function, wherein said one or morecompounds slows or stops the growth of said cancer cell.
 13. The methodof claim 12 wherein said cancer cell is an ovarian cancer cell.
 14. Themethod of claim 13 wherein said ovarian cancer cell is a small cellcarcinoma of the ovary hypercalcemic type (SCCOHT).
 15. The method ofclaim 12 wherein said compound is selected from the group consisting ofa small molecule, a polynucleotide, and a polypeptide.
 16. A method fortreating a patient afflicted with a cancer that is associated with acell exhibiting reduced or undetectable SMARCA4 protein levels and/orprotein function, said method comprising contacting said cancer cellwith one or more compounds that can restore one or more of SMARCA4 geneexpression, SMARCA4 protein level, and/or SMARCA4 protein function,wherein said one or more compounds slows or stops the growth of saidcancer cell.
 17. The method of claim 16 wherein said cancer cell is anovarian cancer cell.
 18. The method of claim 17 wherein said ovariancancer cell is a small cell carcinoma of the ovary hypercalcemic type(SCCOHT).
 19. The method of claim 16 wherein said compound is selectedfrom the group consisting of a small molecule, a polynucleotide, and apolypeptide.
 20. (canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled)24. (canceled)
 25. A diagnostic kit for identifying a cancer cell or fordetecting in a patient a cancer cell that exhibits reduced SMARCA4 geneexpression, reduced SMARCA4 protein function, and/or reduced SMARCA4protein level, which diagnostic kit contains one or more reagents thatcan be used alone or in combination to: (a) detect a mutation in aSMARCA4 gene and/or mRNA; (b) detect a reduction in SMARCA4 mRNA level;(c) detect a reduction in SMARCA4 protein level; and/or (d) detect areduction in SMARCA4 protein functionality, wherein said kit alsocontains positive and negative controls for said identification ordetection.
 26. The diagnostic kit of claim 25 wherein said mutation insaid SMARCA4 gene and/or mRNA is selected from the group consisting ofan insertion mutation, a deletion mutation, a frame shift mutation, asplice site mutation, a nonsense mutation, and a missense mutation. 27.A method for establishing or excluding a diagnosis of small cellcarcinoma of the ovary hypercalcemic type (SCCOHT) in a subject, saidmethod comprising: determining whether a SMARCA4 nucleotide sequencefrom a cancer cell obtained from an ovarian tumor of said subject has amutation as compared to a wild-type SMARCA4 nucleotide sequence,wherein: (a) if said mutation is associated with reduced SMARCA4 geneexpression and/or reduced SMARCA4 protein functionality, the presence ofsaid mutations is predictive of SCCOHT; or (b) if said mutation is notassociated with reduced SMARCA4 gene expression and/or reduced SMARCA4protein functionality, the presence of said mutation is not predictiveof SCCOHT; or (c) if a mutation is absent in said SMARCA4 nucleotidesequence excludes SCCOHT from the diagnosis.
 28. The method of claim 27wherein said mutation in SMARCA4 nucleotide sequence includes a nonsensemutation, a missense mutation, a deletion, an insertion, a frameshiftmutation, and/or a duplication mutation.
 29. The method of claim 27wherein the ascertaining step comprises comparing a SMARCA4 nucleotidesequence in said cancer cell with a SMARCA4 nucleotide sequence in saidcontrol wherein the presence of said mutation in the cancer cell SMARCA4nucleotide sequence indicates SCCOHT; conversely, the absence of saidmutation in the cancer cell SMARCA4 nucleotide sequence excludes SCCOHT.30. The method of claim 27 wherein the ascertaining step comprises:comparing the level of a SMARCA4 gene expression in said cancer cell andto the level of expression in a SMARCA4 gene in said control; wherein areduced level of SMARCA4 gene expression in said cancer cell as comparedto said control indicates SCCOHT; and conversely, the absence of areduced level of SMARCA4 gene expression in said cancer cell as comparedto said control excludes SCCOHT.
 31. A method for detecting whether acancer cell is involved in ovarian cancer, said method comprising:ascertaining a SMARCA4 protein level in said cancer cell in comparisonto a SMARCA4 protein level in a non cancer cell, wherein a reduced levelof SMARCA4 protein in said cancer cell as compared to said non-cancercell indicates that is involved in ovarian cancer.
 32. A method fordetecting whether a cancer cell is involved in ovarian cancer, saidmethod comprising: detecting a carboxy-terminal truncation in a SMARCA4protein in said cancer cell wherein the presence of saidcarboxy-terminal truncation in said SMARCA4 protein indicates that saidcancer cell is involved in ovarian cancer.
 33. A method for detectingwhether a cancer cell is involved in ovarian cancer, said methodcomprising: detecting levels of a SWI/SNF and/or a BAF protein-proteincomplex containing SMARCA4 in said cancer cell in comparison to aswi/snf and/or a baf protein-protein complexes containing SMARCA4 in anon cancer cell, wherein the presence of a reduced level of a SWI/SNFand/or a BAF protein-protein complex containing SMARCA4 in said cancercell indicates that said cancer cell is involved in ovarian cancer. 34.A method for detecting whether a cancer cell is involved in ovariancancer, said method comprising: detecting levels of expression of one ormore SMARCA4 downstream target genes in a cancer cell in comparison to anon-cancer cell, wherein said target gene is selected from the groupconsisting of CDH1, CDH3, EHF, RRAD, and ML-IAP, and wherein a reducedexpression level of said SMARCA4 downstream target gene in said cancercell indicates that said cancer cell is involved in ovarian cancer. 35.A method for treating a patient previously diagnosed with ovarian cancerthat is associated with ovarian cancer cell exhibiting reduced orundetectable SMARCA4 protein levels and/or protein function, said methodcomprising contacting said ovarian cancer cell with one or morecompounds that can restore one or more of SMARCA4 gene expression,SMARCA4 protein level, and/or SMARCA4 protein function, wherein said oneor more compounds slows or stops the growth of said ovarian cancer cell.36. A method for diagnosing and treating SCCOHT in a patient, saidmethod comprising: detecting in a biological sample from a patient oneor more mutations in a SMARCA4 gene and/or an associated regulatorysequence; and either (i) diagnosing the patient with SCCOHT when thepresence of one or more mutations in a SMARCA4 gene and/or associatedregulatory sequence is detected, each of which mutations is known,predicted, or demonstrated to reduce or eliminate at least one ofSMARCA4 gene expression, SMARCA4 protein levels, and SMARCA4 proteinfunction; and (ii) administering an effective amount of one or morecompounds that can restore one or more of SMARCA4 gene expression,SMARCA4 protein level, and/or SMARCA4 protein function to the diagnosedpatient; or (ii) excluding diagnosis of SCCOHT when none of saidmutations is known, predicted or demonstrated to reduce or eliminate atleast one of SMARCA4 gene expression, SMARCA4 protein levels, andSMARCA4 protein function.
 37. A method for diagnosing a predispositionto developing SCCOHT, said method comprising: determining whether aSMARCA4 nucleotide sequence in a biological sample has a mutation ascompared to a wild-type SMARCA4 nucleotide sequence, wherein if saidmutation is present and associated with reduced SMARCA4 gene expressionand/or reduced SMARCA4 protein functionality, the presence of saidmutations is predictive of developing SCCOHT; or if said mutation ispresent and not associated with reduced SMARCA4 gene expression and/orreduced SMARCA4 protein functionality, the presence of said mutation isnot predictive of SCCOHT; or if no mutation in said SMARCA4 nucleotidesequence is present then predisposition to developing SCCOHT is ruledout.